Methods, systems, and compositions for counting nucleic acid molecules

ABSTRACT

Compositions and methods, systems, and kits for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, e.g., due to gene duplication, or to variations from the normal euploid complement of chromosomes, e.g., trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.

The present application is a continuation of PCT/US2020/026456, filedApr. 2, 2020, which claims priority to U.S. Provisional Application Ser.No. 62/828,397, filed Apr. 2, 2019; U.S. Provisional Application Ser.Nos. 62/910,394 and 62/910,397, both filed Oct. 3, 2019; and U.S.Provisional Application Ser. Nos. 62/913,542 and 62/913,543, both filedOct. 10, 2019, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods fordetermining numbers of copies of individual molecules, such as nucleicacid molecules, without digital sequencing. The technologies find use,for example, in analysis of variations in copy numbers of specificnucleic acids sequences that may arise, e.g., from variations inchromosome number, gene copy number, expression level, etc. Thetechnologies find particular application in genetic screening, e.g.,prenatal testing, particularly for non-invasive prenatal testing (NIPT).NIPT is directed to the analysis of cell-free DNA (cfDNA) from a fetusthat circulates in the blood of a woman carrying the fetus in utero.Analysis of cell-free DNA in maternal blood can be used to assess thehealth of the fetus. The technology herein relates to methods, systems,and kits for detecting and quantifying variations in numbers ofmolecules, particularly variations in gene dosage, e.g., due to geneduplication, or to variations from the normal euploid complement ofchromosomes, e.g., trisomy of one or more chromosomes that are normallyfound in diploid pairs.

BACKGROUND OF THE INVENTION

Detection of the presence of, or variations in the numbers of moleculesin a sample is a useful way of characterizing the sample and the sourceof the sample. For example, variations in gene dosage are clinicallysignificant indicators of disease states, e.g., in a subject from whom asample is collected. Variations in gene dosage arise due to errors inDNA replication and can occur in germ line cells, leading to congenitaldefects and even embryonic demise, or in somatic cells, often resultingin cancer. These replication anomalies can cause deletion or duplicationof parts of genes, full-length genes and their surrounding regulatoryregions, megabase-long portions of chromosomes, or entire chromosomes.Analysis of other biomolecules is also clinically important. Forexample, variations in amounts of RNA or protein may indicate changes inexpression of a gene associated with a disease state.

Chromosomal abnormalities can affect either the number or structure ofchromosomes. Conditions wherein cells, tissues, or individuals have oneor more whole chromosomes or segments of chromosomes either absent, orin addition to the normal euploid complement of chromosomes can bereferred to as aneuploidy. Germline replication errors due to chromosomenon-disjunction result in either monosomies (one copy of an autosomalchromosome instead of the usual two or only one sex chromosome) ortrisomies (three copies). Such events, when they do not result inoutright embryonic demise, typically lead to a broad array of disordersoften recognized as syndromes, e.g., trisomy 21 and Down's syndrome,trisomy 18 and Edward's syndrome, and trisomy 13 and Patau's syndrome.Structural chromosome abnormalities affecting parts of chromosomes arisedue to chromosome breakage, and result in deletions, inversions,translocations or duplications of large blocks of genetic material.These events are often as devastating as the gain or loss of the entirechromosome and can lead to such disorders as Prader-Willi syndrome (del15q11-13), retinoblastoma (del 13q14), Cri du chat syndrome (del 5p),and others listed in U.S. Pat. No. 5,888,740, herein incorporated in itsentirety by reference.

Major chromosomal abnormalities are detected in nearly 1 of 140 livebirths and in a much higher fraction of fetuses that do not reach termor are still-born. Hsu (1998) Prenatal diagnosis of chromosomalabnormalities through amniocentesis. In: Milunsky A, editor. GeneticDisorders and the Fetus. 4 ed. Baltimore: The Johns Hopkins UniversityPress. 179-180; Staebler et al. (2005) “Should determination of thekaryotype be systematic for all malformations detected by obstetricalultrasound?” Prenat Diagn 25: 567-573. The most common aneuploidy istrisomy 21 (Down syndrome), which currently occurs in 1 of 730 births.Hsu; Staebler et al. Though less common than trisomy 21, trisomy 18(Edwards Syndrome) and trisomy 13 (Patau syndrome) occur in 1 in 5,500and 1 in 17,200 live births, respectively. Hsu. A large variety ofcongenital defects, growth deficiencies, and intellectual disabilitiesare found in children with chromosomal aneuploidies, and these presentlife-long challenges to families and societies. Jones (2006) Smith'srecognizable patterns of human malformation. Philadelphia: ElsevierSaunders. There are a variety of prenatal tests that can indicateincreased risk for fetal aneuploidy, including invasive diagnostic testssuch as amniocentesis or chorionic villus sampling, which are thecurrent gold standard but are associated with a non-negligible risk offetal loss. American College of Obstetricians and Gynecologists (2007)ACOG Practice Bulletin No. 88, December 2007. Invasive prenatal testingfor aneuploidy. Obstet Gynecol 110: 1459-1467. More reliable,non-invasive tests for fetal aneuploidy have therefore long been sought.The most promising of these are based on the detection of fetal DNA inmaternal plasma. It has been demonstrated that massively parallelsequencing of libraries generated from maternal plasma can reliablydetect chromosome 21 abnormalities. See, e.g., Chiu et al., Noninvasiveprenatal diagnosis of fetal chromosomal aneuploidy by massively parallelgenomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA105:20458-20463 (2008); Fan et al., Noninvasive diagnosis of fetalaneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl AcadSci USA 105: 16266-16271 (2008). See also U.S. Pat. No. 7,888,017.

Quantifying variations in numbers of molecules is not limited to NIPTanalysis, but finds application broadly in analyzing nucleic acids forany purpose, e.g., for characterizing nucleic acids and nucleic acidmixtures, such as nucleic acids indicative of cancer or other disease ina subject, nucleic acids indicative of microbes, e.g., viral andbacterial microbes and mixtures of microbes in a sample, etc.

Current methods for quantifying variations in numbers of molecules, forexample performing aneuploidy screening, that rely on next generationsequencing (NGS) are often time-consuming, expensive, and requireextensive bioinformatics analysis.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods, and systems forthe detection and characterization of samples by counting particularmolecules (e.g., small molecules, haptens, proteins, antibodies, lipids,carbohydrates, and nucleic acids, such as genes or other DNA moleculesor fragments, and/or RNAs, e.g., messenger RNAs, microRNAs and othernon-coding RNAs) that may be represented in the samples. The technologyfinds application, for example, in monitoring gene expression, measuringnon-coding RNA abundance, and in analyzing genetic variations, includingbut not limited to alterations in gene dosage, such as, e.g.,aneuploidy. In preferred embodiments, the technology provides methodsfor detecting and thereby counting single copies of target molecules,including nucleic acids, without the use of “next generation” sequencing(NGS) technologies, such as those described by Chiu et al. and Fan, etal., supra, or on single-molecule amplification technologies that relyon separating amplification reactions for individual target molecules indifferent physically discrete elements, e.g., micro-vessels or emulsiondroplets. While embodiments of the technology provided herein arediscussed in relation particular applications, e.g., measuring DNA, itwill be appreciated that the technology is not limited to theseapplications, and that it is readily adapted to analysis of manydifferent types of molecules, modifications on molecules (e.g.,glycosylation, phosphorylation, methylation), or moieties capable ofbinding to a partner molecule in a specific manner, e.g., antigens withantibodies, nucleic acids with complementary nucleic acids, nucleic acidstructures (e.g., stem-loops, bulged nucleotides, flaps, promotersequences) with proteins that bind such structures, lectins withcarbohydrates, proteins with protein binding partners, proteins withlipids (e.g., SH2 domains with lipids), etc.

In general, these compositions, methods, and systems offer improvedmeans to detect genomic deletions and duplications of various sizes,including complete chromosomes, arms of chromosomes, microscopicdeletions and duplications, submicroscopic deletions and deletions, andsingle nucleotide features, including single nucleotide polymorphisms,deletions, and insertions. In certain embodiments, the methods of thedisclosure can be used to detect sub-chromosomal genetic lesions, e.g.,microdeletions. Exemplary applications of the methods include pediatricand prenatal diagnosis of aneuploidy, testing for product of conceptionor risk of premature abortion, noninvasive prenatal testing (bothqualitative and quantitative genetic testing, such as detectingMendelian disorders, insertions/deletions, and chromosomal imbalances),testing preimplantation genetics, tumor characterization, postnataltesting including cytogenetics, and mutagen effect monitoring.

In some embodiments, the technology herein provides methods forcharacterizing nucleic acid, preferably DNA, more preferably circulatingcell-free DNA (ccfDNA) from blood or plasma, in a sequence-specific andquantitative manner. In preferred embodiments, single copies of the DNAare detected and counted, without polymerase chain reaction or DNAsequencing. Embodiments of the technology provide methods, compositions,and systems for detecting target DNA using methods for amplifyingsignals that are indicative of the presence of the target DNA in thesample. In preferred embodiments, the detectable signal from a singletarget molecule is amplified to such an extent and in such a manner thatthe signal derived from the single target molecule is detectable andidentifiable, in isolation from signal from other targets and from othercopies of the target molecule.

Embodiments of the technology provide methods for counting productsformed by rolling circle replication, e.g., in a rolling circleamplification (RCA) reaction. In some embodiments the technologyprovides methods of counting RCA product molecules formed by replicationfrom circularized nucleic acid probe molecules, e.g., molecularinversion probes (MIPs), including, e.g., padlock probes. Circularizednucleic acid probes may be formed, for example, by hybridization of alinear probe molecule having unique polynucleotide arms designed tohybridize immediately upstream and downstream of a specific targetsequence (or site) in a nucleic acid target, e.g., in an RNA, cfDNA, orgenomic nucleic acid sample and ligating the arms together to form acircularized nucleic acid probe. In some embodiments a MIP probe forms aligatable nick upon hybridization to the nucleic acid target, while insome embodiments, the MIP probe is modified or repaired (e.g., by gapfilling, flap cleavage, etc.) to form a nick prior to ligation. Inembodiments, a number or amount of circularized nucleic acid probesformed in a reaction mixture is indicative of a number or amount oftarget nucleic acids in the reaction mixture.

In embodiments of the technology, a method is provided for countingcircularized nucleic acid probes, comprising:

-   -   a) providing a ligation mixture comprising circularized nucleic        acid probes and linear nucleic acids;    -   b) treating the ligation mixture with at least one exonuclease,        wherein circularized nucleic acid probes are not substrate for        the at least one exonuclease;    -   c) forming a plurality of complexes, each complex comprising an        oligonucleotide primer hybridized to a circularized nucleic acid        probe from the treated ligation mixture;    -   d) detecting formation of the plurality of complexes in a        process comprising:        -   i) extending primers in the complexes in a rolling circle            amplification (RCA) reaction to form RCA products that            comprise primer portions;        -   ii) hybridizing labeled probes to the RCA products, wherein            RCA products with hybridized labeled probes are localized to            a support at dispersed loci, wherein at least a portion of            the RCA products localized at the dispersed loci are            individually detectable by detection of hybridized labeled            probes; and        -   iii) counting RCA products at dispersed loci on the support,            preferably the counting RCA products at dispersed loci on            the support by microscopy.

In some embodiments, the primers are localized at the dispersed lociprior to the extending, while in some embodiments, the primer portionsof the RCA products are localized to the dispersed loci after theextending.

In any of the embodiments described above, embodiments are providedwherein:

-   -   a) the primers or primer portions are bound to one or more        surfaces, preferably covalently linked to the one or more        surfaces, or    -   b) the primers or primer portions are hybridized to capture        oligonucleotides, wherein the capture oligonucleotides are bound        to one or more surfaces, preferably covalently linked to the one        or more surfaces.

In particular embodiments, the primers are bound to the one or moresurfaces, preferably covalently linked to the one or more surfaces, orare hybridized to capture oligonucleotides bound to the one or moresurfaces, preferably covalently linked to the one or more surfaces,before the extending.

In any of the embodiments described above, the technology furtherprovides embodiments wherein the support comprises one or more surfacesselected from a portion of an assay plate, preferably a multi-well assayplate, preferably a glass-bottom assay plate; a portion of a slide; andone or more particles, preferably nanoparticles, wherein the particlesare preferably paramagnetic particles, preferably ferromagneticnanoparticles, preferably iron oxide nanoparticles.

In certain of any of the embodiments described above, the primers arebound to surfaces on particles, preferably covalently linked to surfaceson the particles, and wherein the RCA products with hybridized labeledprobes are localized to dispersed loci by one or more of a magnet,centrifugation, and filtration.

In any one of the embodiments described above, the dispersed loci may bein an irregular dispersal or the dispersed loci may be in an addressablearray.

Any of the embodiments described above comprise embodiments whereinhybridized labeled probes comprise oligonucleotides comprising afluorescent label or a quencher moiety, or both a fluorescent label anda quencher moiety. The technology includes but is not limited toembodiments wherein a plurality of RCA products are hybridized tolabeled probes that all comprise the same label, preferably the samefluorescent label, and embodiments wherein a plurality of RCA productsare hybridized to labeled probes, that comprise two, three, four, five,six, seven or more different labels, preferably two, three, four, five,six, seven, or more different fluorescent labels.

In any of the embodiments above, forming RCA products may compriseextending the primers in the complexes in a reaction mixture comprisingpolyethylene glycol (PEG), preferably at least 2 to 10% (w:v),preferably at least 12%, preferably at least 14%, preferably at least16%, preferably at least 18% to 20% PEG. In any of these embodiments,PEG may have an average molecular weight between 200 and 8000,preferably between 200 and 1000, preferably between 400 and 800,preferably 600.

In any of the embodiments above, forming RCA products may compriseincubating a reaction mixture for an incubation period having abeginning and an end, wherein the reaction mixture is treated by mixingone or more times between the beginning of the incubation period and theend of the incubation period, wherein the mixing preferably comprisesone or more of vortexing, bumping, rocking, tilting, and ultrasonicmixing.

In any of the embodiments above, providing the ligation mixturecomprising circularized nucleic acid probes and linear nucleic acids maycomprise ligating MIP probes, preferably padlock probes, in the presenceof a target nucleic acid target nucleic acid, preferably a targetnucleic acid from a sample, preferably a target nucleic acid from asample, to form the circularized nucleic acid probes. The target nucleicacid is not limited to any particular type of target nucleic acid, anymay comprise, e.g., DNA, RNA, genomic DNA, cfDNA, synthetic DNA, etc.

In any of the embodiments above, the at least one exonuclease maycomprise one or more of Exonuclease I (Exo I, E. coli), ThermolabileExonuclease I; Exonuclease VII (Exo VII, E. coli), Exonuclease T (or“RNase T”) and RecJ_(f), a recombinant fusion protein of E. coli RecJand maltose binding protein (MBP). In any of these embodiments, treatingthe ligation mixture with at least one exonuclease may compriseinactivating the at least one exonuclease, preferably heat-inactivatingthe at least one exonuclease, prior to forming the plurality ofcomplexes.

In embodiments described above, forming RCA products may compriseextending the primers in the complexes in a reaction mixture thatcomprises the labeled probes, and/or may comprise embodiments whereinRCA products are localized at the dispersed loci prior to hybridizingthe labeled probes to the RCA products. In some embodiments, RCAproducts with hybridized labeled probes are treated with graphene oxideprior to counting the RCA products at the dispersed loci.

Any of the embodiments above may comprise embodiments wherein RCAproducts with hybridized labeled probes are treated with one or moredetergents prior to counting the RCA products at the dispersed loci,preferably one or more detergents comprising agents selected fromanionic agents, preferably sodium dodecyl sulfate; sodium laurylsulfate; ammonium lauryl sulfate; cationic agents, preferablybenzalkonium chloride; cetyltrimethylammonium bromide; linearalkylbenzene sulfonates, preferably sodium dodecylbenzene sulfonate;non-ionic agents, preferably a TWEEN detergent selected frompolyoxyethylene (20) sorbitan-monolaurate; -monopalmitate;-monostearate; or -monooleate; a TRITON detergent preferably selectedfrom s polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether,and steroid and steroidal glycosides, preferably saponin or digitonin;and zwitterionic agents, preferably3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); andmixtures of detergent agents, preferably TEEPOL® detergent, comprisingsodium dodecylbenzene sulfonate, and sodium C₁₂-C₁₅ alcohol ethersulfate.

Any of the embodiments above may comprise embodiments wherein thesupport comprises a an organic coating, the coating preferablycomprising a polymeric coating polymerized from surface-modifyingmonomers, wherein the surface-modifying monomers preferably comprise oneor more of dopamine, tannic acid, caffeic acid, pyrogallol, gallic acid,epigallocatechin gallate, and epicatechin gallate monomers, preferablydopamine and tannic acid. In some embodiments, the polymeric coating ishomopolymeric. See, e.g., US 2003/0087338, which is incorporated hereinby reference for all purposes.

Any of the embodiments above may comprise embodiments wherein prior tolocalizing RCA products at the dispersed loci, the primers, primerportions, or capture oligonucleotides comprise one or moreimmobilization moieties, preferably selected from a reactive amine, areactive thiol group, biotin, and a hapten, wherein the immobilizationmoieties are exposed to a surface under conditions wherein theimmobilization moieties interact with the surface to bind the primers,primer portions, or capture oligonucleotides to the surface. In certainembodiments, prior to localizing RCA products at the dispersed loci thesurface comprises at least one of:

-   -   acrylic groups;    -   thiol-containing groups;    -   reactive amine groups;    -   carboxyl groups,    -   streptavidin,    -   antibodies,    -   haptens,    -   carbohydrates,    -   lectins.

Embodiments of the technology provide a method for counting circularizednucleic acid probes, comprising:

-   -   a) providing a ligation mixture comprising circularized nucleic        acid probes and linear nucleic acids;    -   b) forming a plurality of complexes, each complex comprising an        oligonucleotide primer hybridized to a circularized nucleic acid        probe from the ligation mixture, wherein the primer is bound to        a nanoparticle, preferably a paramagnetic nanoparticle;    -   c) detecting formation of the plurality of complexes in a        process comprising:        -   i) extending primers in the complexes in a rolling circle            amplification (RCA) reaction to form RCA products bound to            nanoparticles, wherein at least a portion of the RCA            products on nanoparticles are individually detectable; and        -   iii) counting RCA products on the nanoparticles.

Some embodiments comprise hybridizing labeled probes to the RCAproducts, wherein at least a portion of the RCA products areindividually detectable by detection of hybridized labeled probes. Anyof the embodiments described above comprise embodiments whereinhybridized labeled probes comprise oligonucleotides comprising afluorescent label or a quencher moiety, or both a fluorescent label anda quencher moiety. The technology includes but is not limited toembodiments wherein a plurality of RCA products are hybridized tolabeled probes that all comprise the same label, preferably the samefluorescent label, and embodiments wherein a plurality of RCA productsare hybridized to labeled probes, that comprise two, three, four, five,six, seven or more different labels, preferably two, three, four, five,six, seven, or more different fluorescent labels.

In any of the embodiments wherein the primer is bound to a nanoparticle,the method comprises embodiments wherein the nanoparticles areparamagnetic nanoparticles, preferably iron oxide nanoparticles. Inembodiments the nanoparticles have an average diameter of less thanabout 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm,200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm,10 nm, 5 nm, or 1 nm in diameter, wherein the nanoparticles arepreferably from 1 to 50 nm, preferably from 5 to 20 nm average diameter.In some embodiments, the nanoparticles comprise an inorganic core ofabout 2.5 to about 55 nm diameter, and an organic coating, the organiccoating preferably having an overall thickness of about 3 to 5 nm.Preferably the nanoparticles are predominantly spheroid or spherical,and in certain embodiments, the nanoparticles are essentially uniform indiameter.

In any of the embodiments wherein the primer is bound to a nanoparticleinclude embodiments wherein prior to binding primers, the nanoparticleshave a surface comprising reactive groups, the reactive groupspreferably comprising at least one of:

-   -   acrylic groups;    -   thiol-containing groups;    -   reactive amine groups;    -   carboxyl groups,

wherein the primers comprise reactive groups suitable for formingcovalent bonds with reactive groups on the surface of the nanoparticles,and wherein the primers and the nanoparticles are treated together underconditions wherein the primers are covalently linked to thenanoparticles.

In any of the embodiments wherein the primer is bound to a nanoparticle,counting RCA products on nanoparticles may comprise at least one offluorescence microscopy, flow cytometry, and nanopore sensing.

In any of the embodiments wherein the primer is bound to a nanoparticle,counting RCA products on nanoparticles may comprise localizing RCAproducts to a support at dispersed loci wherein at least a portion ofthe RCA products localized at the dispersed loci are individuallydetectable by detection of hybridized labeled probes and counting RCAproducts at dispersed loci on the support. In preferred embodiments, RCAproducts with hybridized labeled probes are localized to dispersed lociby one or more of a magnet, centrifugation, and filtration.

Any of the embodiments wherein the primer is bound to a nanoparticleinclude embodiments wherein prior to forming the plurality of complexes,the ligation mixture is treated with at least one exonuclease, whereincircularized nucleic acid probes are not substrate for the at least oneexonuclease. In preferred embodiments, the at least one exonucleasecomprises at least one exonuclease selected from Rec Jf, Exo VII, Exo T,and Thermolabile Exo I.

Embodiments of the technology provide a composition comprising aplurality of complexes bound to a surface of an organic coating on oneor more supports, wherein the one or more supports preferably compriseone or more of an assay plate, preferably a glass-bottom assay plate,and a nanoparticle, preferably a paramagnetic nanoparticle, morepreferably a ferromagnetic nanoparticle, preferably an iron oxidenanoparticle, each complex comprising an oligonucleotide primerhybridized to a circularized nucleic acid probe, wherein the primer isbound to the surface of the organic coating on the support, and areaction mixture comprising:

-   -   Phi29 DNA polymerase, preferably at least 0.2 units per μL,        preferably at least 0.8 units per μL of Phi29 DNA polymerase;    -   a buffer;    -   a mixture of dNTPs, preferably at least 400 μM, preferably at        least 600 μM, more preferably at least 800 μM total dNTPs;    -   PEG, preferably at least 2 to 10% (w:v), preferably at least        12%, preferably at least 14%, preferably at least 16%,        preferably at least 18% to 20% PEG.

In some embodiments, the PEG has an average molecular weight between 200and 8000, preferably between 200 and 1000, preferably between 400 and800, preferably 600.

Embodiments include any of the compositions described above, wherein thereaction mixture further comprises at least one labeled probe,preferably a fluorescently labeled probe, more preferably a molecularbeacon probe, preferably least 100 nM of labeled probe, more preferablyat least 1000 nM of labeled probe.

Embodiments of the technology further provide a composition comprising aplurality of RCA products bound to a surface of an organic coating onone or more supports, wherein the one or more supports preferablycomprise one or more of an assay plate, preferably a glass-bottom assayplate, and a nanoparticle, preferably a paramagnetic nanoparticle, morepreferably a ferromagnetic nanoparticle, preferably an iron oxidenanoparticle, each RCA product comprising a primer portion bound to thesurface of the organic coating on the support, and a buffer comprisingMg′, the solution further comprising:

-   -   one or more labeled probes hybridized to RCA products; and    -   one or more of:        -   graphene oxide;        -   one or more detergents.

Embodiments of such compositions include embodiments wherein the labeledprobes comprise fluorescent labels and embodiments wherein the labeledprobes comprise quencher moieties. In some embodiments, a plurality ofRCA products are hybridized to labeled probes that all comprise the samelabel, preferably the same fluorescent label, while in some embodiments,a plurality of RCA products are hybridized to a set of labeled probesthat all comprise a label, wherein the set of labeled probes comprisestwo, three, four, five, six, seven, or more different labels, preferablytwo, three, four, five, six, seven, or more different fluorescentlabels.

Embodiments include any of the compositions above in which the one ormore detergents comprise agents selected from anionic agents, preferablysodium dodecyl sulfate; sodium lauryl sulfate; ammonium lauryl sulfate;cationic agents, preferably benzalkonium chloride;cetyltrimethylammonium bromide; linear alkylbenzene sulfonates,preferably sodium dodecylbenzene sulfonate; non-ionic agents, preferablya TWEEN detergent selected from polyoxyethylene (20)sorbitan-monolaurate; -monopalmitate; -monostearate; or -monooleate; aTRITON detergent preferably selected from s polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether, and steroid and steroidalglycosides, preferably saponin or digitonin; and zwitterionic agents,preferably 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS); and mixtures of detergent agents, preferably TEEPOL® detergent,comprising sodium dodecylbenzene sulfonate, and sodium C₁₂-C₁₅ alcoholether sulfate.

Any of the embodiments above include embodiments of the compositionwherein the organic coating is a polymeric coating polymerized fromsurface-modifying monomers, wherein the surface-modifying monomerspreferably comprise one or more of dopamine, tannic acid, caffeic acid,pyrogallol, gallic acid, epigallocatechin gallate, and epicatechingallate monomers, preferably dopamine and tannic acid. In someembodiments, the polymeric coating is homopolymeric.

Any of the embodiments above include embodiments of the compositionwherein the solution comprising a labeled probe comprises afluorescently labeled probe, preferably a molecular beacon probe,preferably more than 100 nM of labeled probe, preferably at least 1000nM of labeled probe, and/or wherein the buffer comprising Mg′ is a Phi29DNA polymerase buffer.

Embodiments of the technology comprise systems, for example, a systemcomprising:

-   -   i) a plurality of complexes bound to a surface of an organic        coating on one or more supports, wherein the one or more        supports preferably comprise one or more of an assay plate,        preferably a glass-bottom assay plate, and a nanoparticle,        preferably a paramagnetic nanoparticle, more preferably a        ferromagnetic nanoparticle, preferably an iron oxide        nanoparticle, each complex comprising an oligonucleotide primer        hybridized to a circularized nucleic acid probe, wherein the        primer is bound to the surface of the organic coating on the        support;    -   ii) DNA polymerase, preferably Phi29 DNA polymerase;    -   iii) one or more labeled probes, preferably fluorescently        labeled probes.

In some embodiments, a system further comprises one or more of:

-   -   iv) a buffer comprising Mg++, preferably a buffer comprising        MgCl₂, preferably a Phi29 DNA polymerase buffer;    -   v) PEG, preferably PEG having an average molecular weight        between 200 and 8000, preferably between 200 and 1000,        preferably between 400 and 800, preferably 600;    -   vi) one or more detergents, preferably one or more detergents        comprising agents selected from anionic agents, preferably        sodium dodecyl sulfate; sodium lauryl sulfate; ammonium lauryl        sulfate; cationic agents, preferably benzalkonium chloride;        cetyltrimethylammonium bromide; linear alkylbenzene sulfonates,        preferably sodium dodecylbenzene sulfonate; non-ionic agents,        preferably a TWEEN detergent selected from polyoxyethylene (20)        sorbitan-monolaurate; -monopalmitate; -monostearate; or        -monooleate; a TRITON detergent preferably selected from s        polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether,        and steroid and steroidal glycosides, preferably saponin or        digitonin; and zwitterionic agents, preferably        3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate        (CHAPS); and mixtures of detergent agents, preferably TEEPOL®        detergent, comprising sodium dodecylbenzene sulfonate, and        sodium C₁₂-C₁₅ alcohol ether sulfate;    -   vii) as solution comprising dNTPs; and    -   viii) graphene oxide.

In some embodiments, the organic coating is a polymeric coatingpolymerized from surface-modifying monomers, wherein thesurface-modifying monomers preferably comprise one or more of dopamine,tannic acid, caffeic acid, pyrogallol, gallic acid, epigallocatechingallate, and epicatechin gallate monomers, preferably dopamine andtannic acid, and in some embodiments, the polymeric coating ishomopolymeric.

In some embodiments, the support comprises a first surface. In someembodiments, the support, or the first surface of the support, comprisesa solid or a porous material. In embodiments, the support or the firstsurface of the support can comprise ceramic, silanized material, metal,polymer, stone, paper, fabric, a carbon material, or a combinationthereof. As used herein, the term “carbon materials” refer to elementalcarbon materials, such as graphite, carbon fiber, carbon nanotube,graphene, carbon black, activated carbon, fullerene and diamond. Inembodiments, the silanized material is quartz or glass, such as a glassslide, a glass bead, or a glass plate, such as the surface of a glassbottom assay plate. In some embodiments, the polymer is an organicpolymer. In some embodiments, the polymer is a fluropolymer (e.g.,Teflon®) or an organic polymer. Suitable organic polymers include butare not limited to polyesters (e.g., polyethylene terephthalate orpolyethylene naphthalates), polyacrylates (e.g., polymethyl methacrylateor “PMMA”), poly(vinyl acetate) (“PVAC”), poly(vinyl butyral) (“PVB”),poly(ethyl acrylate) (“PEA”), poly(diphenoxyphosphazene) (“PDPP”),polycarbonate (“PC”), polypropylene (“PP”), high density polyethylene(“HDPE”), low density polyethylene (“LDPE”), polysulfone (“PS”),polyether sulfone (“PES”), polyurethane (“PUR”), polyamide (“PA”),poly(dimethylsiloxane) (“PDMS”), polyvinyl chloride (“PVC”),polyvinylidene fluoride (“PY dF”), polystyrene (“PSy”) and polyethylenesulfide; cellulose derivatives, polyimide, polyimide benzoxazole,polybenzoxazole, poly(glycolic acid), poly(lactic acid),poly(lactic-co-glycolic acid).

In some embodiments, the technology provides a method of preparing asupport, such as a solid support. In some embodiments, the methodcomprises a) providing a first surface (or substrate; or substratehaving a first surface); b) modifying the first surface with one or moresurface modifying agent(s) (SMA(s)); c) thereby providing a supportcomprising a second surface (or coating]). In some embodiments, thesecond surface coats at least a portion of the first surface. In someembodiments, the second surface (or coating) comprises functional groupscapable of forming complexes with one or more analytes. Thus, in someembodiments, the method thereby provides a support referred to herein asa “surface functionalized substrate” (SFS). In some embodiments, thefunctional groups capable of complexing with the one or more analytes isan amine group (e.g., a primary, secondary, tertiary or quaternaryamine), a carboxylate or carboxylic acid group, or a combinationthereof.

In some further embodiments, the modification of the first surface withthe one or more SMAs comprises contacting the first surface with amixture comprising a carrier and the one or more SMAs. In someembodiments, the mixture further comprises one or more initiators, forexample, one or more initiators of polymerization. In some embodiments,the mixture is a solution or a suspension. Thus, in some embodiments,the modification of the first surface comprises contacting the firstsurface with a mixture comprising one or more SMAs, one or moreinitiators, and a carrier. In some further embodiments, the carrier is aliquid carrier, and the mixture is a solution or suspension. In yetfurther embodiments, the liquid carrier is an aqueous vehicle, and themixture is an aqueous solution or suspension. Thus, in some moreparticular embodiments, the modification of the first surface comprisescontacting the first surface with a mixture comprising a carrier, one ormore SMAs and one or more initiators; optionally, the mixture is anaqueous solution or suspension. In some preferred embodiments, themixture is an aqueous solution.

In some embodiments, at least one of the one or more SMAs is a vinylmonomer. In some embodiments, the vinyl monomer can comprise acrylamidessuch as acrylamide, methacrylamide, dialkylaminoalkyl acrylamides, suchas dimethylaminoethyl acrylamide and methacrylamide, acrylates such asacrylic acid and methacrylic acid, dialkylaminoalkyl acrylates, such asdimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, vinylpyridine, methyl vinyl pyridine, vinyl pyrrolidone, amino styrene suchas p-dimethylaminomethyl styrene, vinyl sulfuric acid, trimethylammonium ethyl acrylate (chloride), or a combination thereof. In someembodiments, the vinyl monomer can comprise a carboxylic acid group, anamine group, or both. In some embodiments, the vinyl monomer can bewater or alcohol soluble. In embodiments, the vinyl monomer can comprisean acrylate monomer. In embodiments, the acrylate monomer can comprise acarboxylic acid, an amine, or a combination thereof. In embodiments, theacrylate monomer comprises acrylic acid, methacrylate, ethyl acrylate,propyl acrylate, a butyl acrylate, or a combination thereof. In someembodiments, the acrylate monomer comprises 2-aminoethyl methacrylate(AEMA), acrylic acid (AA), or a combination thereof.

In some embodiments, at least one of the one or more SMAs is a phenolmonomer (i.e., a monomer comprising a phenol group). The phenol monomercan comprise two or more phenolic hydroxyl groups. For example, thephenol monomer can comprise a galloyl group, a catechol group, or acombination thereof. As used herein, the term “galloyl group” comprisesa structure:

As used herein, the term “catechol group” comprises a1,2-dihydroxybenzene. The galloyl group and catechol group used hereincan be further substituted. In embodiments, the phenol monomer cancomprise dopamine, tannic acid, caffeic acid, pyrogallol, gallic acid,epigallocatechin gallate, epicatechin gallate, epigallocatechin, or acombination thereof. In embodiments, the phenol monomer can comprisedopamine. In embodiments, the phenol monomer can comprise tannic acid.

In some embodiments, the method of modifying the first surface comprisespolymerizing the one or more SMAs in the presence of the first surface.Thus, in some embodiments, the method of modifying the first surfacecomprises contacting the first surface with a mixture comprising acarrier and one or more SMAs, wherein the one or more SMAs polymerizesin the presence of the first surface, thereby providing the secondsurface. In some more particular embodiments, the mixture furthercomprises one or more initiators, wherein the initiator(s) initiatepolymerization of the one or more SMAs. In some embodiments, the mixturecomprises one SMA and the polymerization provides a homopolymer. Inother embodiments, the mixture comprises at least two SMAs and thepolymerization provides a copolymer. The homopolymer or the copolymerforms or is deposited on the first surface, thereby providing the secondsurface. In some embodiments, the second surface coats at least aportion of the first surface. In some embodiments, the second surfacecomprises functional groups capable of forming complexes with one ormore analytes. Thus, in some embodiments, the method thereby provides asupport which is a surface functionalized substrate (SFS). In someembodiments, the functional groups capable of complexing with the one ormore analytes is an amine group (e.g., a primary, secondary, tertiary orquaternary amine), a carboxylate or carboxylic acid group, or acombination thereof.

In some embodiments, the polymerization or copolymerization of theSMA(s) can be performed in the presence of an initiator. The initiatorcan initiate polymerization, such as the homopolymerization orcopolymerization of monomers. In some embodiments, the initiator caninitiate the polymerization via a radical polymerization. Inembodiments, the initiator can comprise an oxidant, a base, or acombination thereof. In embodiments, the initiator can comprisehalogens, azo compounds, organic peroxides, inorganic peroxides, or acombination thereof. In embodiments, the initiator can comprise ammoniumpersulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), or acombination thereof. In embodiments, the initiator can initiatepolymerization thermally, under ambient conditions or a combinationthereof. In some embodiments, SMAs comprise photopolymers andpolymerization is initiated by light, e.g., from a halogen, argon, xenonor LED light source.

In some more particular embodiments, the method of preparing the supportcomprises a) providing a substrate having a first surface; b) modifyingthe first surface by contacting the first surface with a mixturecomprising a carrier, a first SMA which is dopamine, a second SMA whichis AEMA, and one or more initiators; c) thereby providing a supportcomprising a second surface, wherein the second surface comprises acopolymer derived from the dopamine and the AEMA, and wherein thesupport is a surface functionalized substrate. In some embodiments, thesecond surface coats at least a portion of the first surface. In someembodiments, the second surface comprises functional groups capable offorming complexes with one or more analytes. In some embodiments, thefunctional groups capable of complexing with the one or more analytes isan amine group, a carboxylate or carboxylic acid group, or a combinationthereof. In some embodiments, the initiator is ammonium persulfate,TEMED, or a combination thereof. In some other embodiments, theinitiator is a photoinitiator. In some embodiments, the mixture is anaqueous solution. In some embodiments, the first surface is a silanizedsurface, such as glass. In some other embodiments, the first surfacecomprises an organic polymer, such as polystyrene.

In some more particular embodiments, the method of preparing the supportcomprises a) providing a substrate having a first surface; b) modifyingthe first surface by contacting the first surface with a mixturecomprising a carrier, a first SMA which is dopamine, a second SMA whichis acrylic acid, and one or more initiators; c) thereby providing asupport comprising a second surface, wherein the second surfacecomprises a copolymer derived from the dopamine and the acrylic acid,and wherein the support is a surface functionalized substrate. In someembodiments, the second surface coats at least a portion of the firstsurface. In some embodiments, the second surface comprises functionalgroups capable of forming complexes with one or more analytes. In someembodiments, the functional groups capable of complexing with the one ormore analytes is an amine group, a carboxylate or carboxylic acid group,or a combination thereof. In some embodiments, the initiator is ammoniumpersulfate, TEMED, or a combination thereof. In some other embodiments,the initiator is a photoinitiator. In some embodiments, the mixture isan aqueous solution. In some embodiments, the first surface is asilanized surface, such as glass. In some other embodiments, the firstsurface comprises an organic polymer, such as polystyrene.

In some more particular embodiments, the method of preparing the supportcomprises a) providing a substrate having a first surface; b) modifyingthe first surface by contacting the first surface with a mixturecomprising a carrier, a first SMA which is tannic acid, a second SMAwhich is AEMA, and one or more initiators; c) thereby providing asupport comprising a second surface, wherein the second surfacecomprises a copolymer derived from the tannic acid and the AEMA, andwherein the support is a surface functionalized substrate. In someembodiments, the second surface coats at least a portion of the firstsurface. In some embodiments, the second surface comprises functionalgroups capable of forming complexes with one or more analytes. In someembodiments, the functional groups capable of complexing with the one ormore analytes is an amine group, a carboxylate or carboxylic acid group,or a combination thereof. In some embodiments, the initiator is ammoniumpersulfate, TEMED, or a combination thereof. In some other embodiments,the initiator is a photoinitiator. In some embodiments, the mixture isan aqueous solution. In some embodiments, the first surface is asilanized surface, such as glass. In some other embodiments, the firstsurface comprises an organic polymer, such as polystyrene.

In some more particular embodiments, the method of preparing the supportcomprises a) providing a substrate having a first surface; b) modifyingthe first surface by contacting the first surface with a mixturecomprising a carrier, a first SMA which is tannic acid, a second SMAwhich is acrylic acid, and one or more initiators; c) thereby providinga support comprising a second surface, wherein the second surfacecomprises a copolymer derived from the tannic acid and the acrylic acid,and wherein the support is a surface functionalized substrate. In someembodiments, the second surface coats at least a portion of the firstsurface. In some embodiments, the second surface comprises functionalgroups capable of forming complexes with one or more analytes. In someembodiments, the functional groups capable of complexing with the one ormore analytes is an amine group, a carboxylate or carboxylic acid group,or a combination thereof. In some embodiments, the initiator is ammoniumpersulfate, TEMED, or a combination thereof. In some other embodiments,the initiator is a photoinitiator. In some embodiments, the mixture isan aqueous solution. In some embodiments, the first surface is asilanized surface, such as glass. In some other embodiments, the firstsurface comprises an organic polymer, such as polystyrene.

In some embodiments, the technology provides a method for countingtarget molecules on a support, comprising: a) providing a first surface;b) modifying the first surface with at least one SMA to provide asurface functionalized substrate (SFS); optionally, the SFS comprisesfunctional groups selected from at least one of carboxylate, carboxylicacid and amine groups; c) contacting the SFS with one or more analytes;d) thereby forming a plurality of complexes between the functionalgroups on the SFS and the one or more analytes; and e) counting theplurality of complexes. In some embodiments, the first surface (orsubstrate) is a silanized surface. In some embodiments, the silanizedsurface is glass, while in some embodiments, the surface is unsilanizedglass. In certain preferred embodiments, the silanized surface comprisesa surface treated with 3-aminopropyltriethoxysilane or3-(trimethoxysilyl) propyl methacrylate. See, e.g., WO 2019/195346 A1 toSekedat, et al., Methods, Systems, and Compositions for Counting NucleicAcids (2019), which is incorporated herein by reference in its entirety,for all purposes. In some embodiments, the one or more analytescomprises at least one of an RCA product comprising a plurality ofhybridized labeled probes and a double-stranded scaffold productcomprising a plurality of concatemerized labeled scaffoldoligonucleotides, wherein formation of a complex is indicative of thepresence of a target molecule on the glass surface, and wherein formingsaid plurality of complexes comprises exposing the glass surface to asolution comprising graphene oxide. The surfaces are not limited to anyparticular format. For example, in any of the embodiments of describedabove, the support may comprise a surface in an assay plate, preferablya glass-bottom assay plate. In some embodiments, the assay plate is amulti-well assay plate, preferably a microtiter plate.

In some embodiments of the technology, the primer of any of theembodiments described above is bound directly to the support, preferablycovalently linked to the support. For example, in some embodiments, theprimer comprises a biotin moiety and the support comprises avidin,preferably streptavidin. In certain embodiments, the complex orcomplexes comprise an antibody bound to an antigen or hapten, and insome embodiments, the complex comprises an antigen or hapten bounddirectly to the support. In certain embodiments, the antigen or haptenis covalently attached to the support. In some embodiments, the primeris covalently linked to a support by conjugation of an amide bondbetween an amine and carboxylic acid.

In any of the embodiments described herein, forming a complex orplurality of complexes may comprise exposing the support to a solutioncomprising a crowding agent. In some embodiments, the crowding agentcomprises polyethylene glycol (PEG), preferably at least 2 to 10% (w:v),preferably at least 12%, preferably at least 14%, preferably at least16%, preferably at least 18% to 20% PEG. In certain preferredembodiments, the PEG has an average molecular weight between 200 and8000, preferably between 200 and 1000, preferably between 400 and 800,preferably 600.

In any of the embodiments described above, forming a complex orplurality of complexes may comprise a step of exposing the support to asolution comprising graphene oxide. In preferred embodiments, thesupport is exposed to graphene oxide prior to step detecting hybridizedlabeled probe. In particularly preferred embodiments, the support isexposed to a solution that comprises a mixture of labeled probe andgraphene oxide. In some embodiments, the support or the glass surfaceexposed to a solution comprising graphene oxide is washed with asolution comprising one or more detergents prior to the detecting orcounting. In certain preferred embodiments, the one or more detergentscomprises Tween 20.

In any of the embodiments described above, forming a complex orplurality of complexes may comprise comprising a step of exposing thesupport to a solution comprising one or more detergents or surfactants.In preferred embodiments, the support is exposed to a solutioncomprising one or more detergents or surfactants prior to a step ofdetecting hybridized labeled probe. In certain embodiments, the supportis exposed to a solution that comprises a mixture of labeled probe andone or more detergents or surfactants. In some embodiments, the supportor the glass surface is washed with a solution comprising one or moredetergents or surfactants. In some embodiments, the detergent comprisesan agent selected from anionic agents (e.g., sodium dodecyl sulfate;sodium lauryl sulfate; ammonium lauryl sulfate), cationic agents (e.g.,benzalkonium chloride; cetyltrimethylammonium bromide; linearalkylbenzene sulfonates, such as sodium dodecylbenzene sulfonate),non-ionic agents (e.g., a TWEEN detergent, such as polyoxyethylene (20)sorbitan-monolaurate; -monopalmitate; -monostearate; or -monooleate; aTRITON, such as polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether, or TRITON X-100; steroid and steroidal glycosides such as saponinand digitonin), and zwitterionic agents (e.g., CHAPS, which is3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), mixtures ofdetergent agents (e.g., TEEPOL® 610 S detergent, comprising sodiumdodecylbenzene sulfonate, sodium C₁₂-C₁₅ alcohol ether sulfate), or amixture thereof.

The technology finds use in detecting many different kinds of molecules,including, e.g., molecules as depicted schematically in FIG. 38. In someembodiments, a target molecule comprises nucleic acid, preferably DNAfrom a sample from a subject, preferably a blood or blood productsample. In certain preferred embodiments, the DNA is cell-free DNA froma blood or blood product sample, including but not limited to venousblood, menstrual blood, or other sources of blood and blood productsthat may be drawn from a body, collected from a tissue of a body, in abody, expelled or issued, etc., from a body of a subject. In someembodiments, the cell-free DNA comprises maternal and/or fetal DNA froma maternal blood sample.

Any of the embodiments described herein may comprise forming an RCAproduct in a process comprises extending a primer on a circularizednucleic acid probe in a reaction mixture. Preferably the reactionmixture comprises at least 0.2 units per μL, preferably at least 0.8units per μL of Phi29 DNA polymerase and at least 400 μM, preferably atleast 600 μM, more preferably at least 800 μM total dNTPs. In someembodiments, forming an RCA product comprising a plurality of hybridizedlabeled probes comprises forming the RCA product in a reaction mixturethat further comprises more than 10 nM fluorescently-labeledoligonucleotide, e.g., a molecular beacon probe, preferably at least 100nM fluorescently-labeled oligonucleotides probe, preferably at least1000 nM fluorophore-labeled probe in the reaction mixture. In someembodiments, forming an RCA product comprising a plurality of hybridizedlabeled probes comprises forming the RCA product in a reaction mixturethat does not comprise labeled probe, then treating the RCA product onthe support with a solution that comprises one more labeled probes,preferably a solution that comprises Mg⁺⁺, preferably MgCl₂. In someembodiments, RCA product is removed from the reaction mixture, and insome embodiments washed, e.g., with a buffer, prior to treatment withthe solution comprising one or more labeled probes.

In any of the embodiments described herein, complexes immobilized on asurface may comprise at least one polypeptide, e.g., an antibody, alectin, and/or they may comprise at least one specifically-bindablemolecule selected from a hapten, a carbohydrate, and a lipid.

In some embodiments of the technology, forming an RCA product comprisesincubating the reaction mixture at least 37° C., preferably at least 42°C., preferably at least 45° C. In certain embodiments, the reactionmixture comprises PEG, preferably at least 2 to 10% (w:v), preferably atleast 12%, preferably at least 14%, preferably at least 16%, preferablyat least 18% to 20% PEG.

The technology also provides compositions related to practice of themethods. In some embodiments, the technology provides a compositioncomprising a silanized surface or non-silanized surface, preferably asurface modified using one or more surface modifying agents to provide asecond surface bound to a plurality of complexes, each comprising anoligonucleotide primer hybridized to a circularized nucleic acid probe,wherein the primer is localized to a support, and a reaction mixturecomprising at least 0.1 units per μL, preferably at least 0.2 to 0.8units per μL of Phi29 DNA polymerase; a buffer; at least 400 μM,preferably at least 600 μM, more preferably at least 800 μM total dNTP;and PEG, preferably at least 2 to 20% (w:v), preferably 12 to 18%,preferably 14 to 16%, preferably 15% PEG. In some embodiments, the PEGhas an average molecular weight of between 200 and 8000, preferablybetween 200 and 1000, preferably between 400 and 800, preferably 600. Insome embodiments, the reaction mixture further comprises at least 10 nMfluorescently labeled oligonucleotide, e.g., molecular beacon probe,preferably at least 100 nM fluorescently labeled oligonucleotide,preferably at least 1000 nM fluorescently labeled oligonucleotide. Insome embodiments, RCA product is removed from the reaction mixture, andin some embodiments washed, e.g., with a buffer, prior to treatment withthe solution comprising one or more labeled probes.

In some embodiments of the composition, the primers are localized to thesupport in an irregular dispersal, while in some embodiments, theprimers are localized to the support in an addressable array. In certainembodiments, the primer is covalently linked to the support, while insome embodiments, wherein the primer comprises a biotin moiety and thesupport comprises avidin, preferably streptavidin. In other embodiments,the primer is covalently bound to a bead or particle, preferably a smallnanoparticle, more preferably a paramagnetic small nanoparticle, and thenanoparticle-bound primer is localized to a surface by an application offorce, e.g., with a magnet or centrifuge In some embodiments, thecomplexes comprise an antibody bound to an antigen or hapten and in someembodiments, the complexes comprise an antigen or hapten bound directlyto the support. In some embodiments, the antigen or hapten is covalentlyattached to the support.

In some embodiments of the compositions herein, complexes comprise atleast one polypeptide. In some preferred embodiments, the at least onepolypeptide comprises an antibody or a lectin. In some embodiments, thecomplexes comprise at least one specifically-bindable molecule selectedfrom a hapten, a carbohydrate, and a lipid.

Embodiments of the composition described above may comprise a silanizedsurface bound to a plurality of complexes each comprising an RCA productcomprising a plurality of hybridized labeled probes, and a solutioncomprising graphene oxide.

In some embodiments, the silanized surface is glass. In some preferredembodiments, the silanized surface comprises a surface, preferably aglass surface, treated with 3-aminopropyltriethoxysilane or3-(trimethoxysilyl) propyl methacrylate. In some embodiments, thesurface, preferably a glass surface is not silanized. In certainpreferred embodiments, the surface comprises a polymeric coating formedby polymerization of one or more monomers, including but not limited toe.g., tannic acid, acrylic acid, dopamine, etc. In preferredembodiments, the support comprises a surface comprising polytannic acidor polydopamine.

In some embodiments, the solution comprising graphene oxide furthercomprises a fluorescently labeled probe, e.g., a molecular beacon probe,preferably more than 10 nM of fluorescently labeled probe, preferably atleast 100 nM fluorescently labeled probe, preferably at least 1000 nMfluorescently labeled probe.

In some embodiments of the composition, the solution comprising grapheneoxide comprises a buffer solution comprising MgCl₂. In certainembodiments, the buffer comprising MgCl₂ is a Phi29 DNA polymerasebuffer.

The technology provided herein is not limited to any particular use orapplication. In some embodiments, the technology finds use in analysisof chromosomal aberrations, e.g., aneuploidy, preferably in the contextof non-invasive prenatal testing. For example, some embodiments ofapplications of the technology comprise obtaining a maternal sample thatcomprises both maternal and fetal genetic material, and measuring aplurality of target nucleic acids, wherein the target nucleic acidscomprise specific sequences associated with a first chromosome, whereinthe first chromosome is suspected of being variant (e.g., in gene dosageor chromosome count) in the fetal material, and wherein the targetnucleic acid further comprises specific sequences associated with asecond chromosome, which is not suspected of being variant in the fetalmaterial. The method comprises analyzing an amount of the target nucleicacids associated with the first chromosome and the amount of targetnucleic acids associated with the second chromosome in the sample todetermine whether the amount of the target nucleic acids associated withthe first chromosome differs sufficiently from the amount the targetnucleic acid associated with the second chromosome to indicate achromosomal or gene dosage variant in the fetus. In preferredembodiments, the target nucleic acids associated the first and secondchromosomes are present in both the maternal and fetal genetic materialand are the maternal and fetal nucleic acids the assay is not specificfor one over the other. In preferred embodiments, the maternal sample iscell-free DNA from maternal blood. Statistical methods for analyzingchromosomal aberrations based on measuring amounts of DNA in a sample,including determining aberrations in the fetal DNA when the fetal DNA isa small fraction of the total DNA in a maternal sample, are known in theart. See, e.g., U.S. Pat. No. 6,100,029, which is incorporated herein byreference.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

The transitional phrase “consisting essentially of” as used in claims inthe present application limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention, as discussed inIn re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). Forexample, a composition “consisting essentially of” recited elements maycontain an unrecited contaminant at a level such that, though present,the contaminant does not alter the function of the recited compositionas compared to a pure composition, i.e., a composition “consisting of”the recited components.

As used herein, the terms “subject” and “patient” refer to any organismsincluding plants, microorganisms and animals (e.g., mammals such asdogs, cats, livestock, and humans).

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin. Biological samples may beanimal, including human, fluid, solid (e.g., stool) or tissue, as wellas liquid and solid food and feed products and ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from all of the various families of domesticanimals, as well as feral or wild animals, including, but not limitedto, such animals as canines, felines, ungulates, bear, fish, lagomorphs,rodents, marsupials, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

The term “target” as used herein refers to a molecule sought to besorted out from other molecules for assessment, measurement, or othercharacterization. For example, a target nucleic acid may be sorted fromother nucleic acids in a sample, e.g., by probe binding, amplification,isolation, capture, etc. When used in reference to a hybridization-baseddetection, e.g., polymerase chain reaction, “target” refers to theregion of nucleic acid bounded by the primers used for polymerase chainreaction, while when used in an assay in which target DNA is notamplified, e.g., in capture by molecular inversion probes (MIPS), atarget comprises the site bounded by the hybridization of thetarget-specific arms of the MIP, such that the MIP can be ligated andthe presence of the target nucleic acid can be detected.

The term “source of target nucleic acid” refers to any sample thatcontains nucleic acids (RNA or DNA). Particularly preferred sources oftarget nucleic acids are biological samples including, but not limitedto blood, plasma, serum, saliva, urine, feces, gastrointestinal fluid,cerebral spinal fluid, pleural fluid, milk, lymph, sputum, and semen.

The term “gene dosage” as used herein refers to the copy number of agene, a genic region, a chromosome, or fragments or portions thereof.Normal individuals carry two copies of most genes or genic regions, oneon each of two chromosomes. However, there are certain exceptions, e.g.,when genes or genic regions reside on the X or Y chromosomes, or whengenes sequences are present in pseudogenes.

The term “aneuploidy” as used herein refers to conditions wherein cells,tissues, or individuals have one or more whole chromosomes or segmentsof chromosomes either absent, or in addition to the normal euploidcomplement of chromosomes.

As used herein, the “sensitivity” of a given assay (or set of assaysused together) refers to the percentage of samples that report aparticular form or variant, e.g., a mutation, gene duplication,chromosome duplication, above a threshold value that distinguishesbetween samples exhibiting a variant phenotype (e.g., cancerous cells,aneuploidy) and samples exhibiting a normal or wild-type phenotype(e.g., non-cancerous cells, euploidy). In some embodiments, a “positive”is defined as a clinically-confirmed variant that reports an assayresult associated with the presence of the disease or condition to bedetected, and a false negative is defined as a clinically-confirmedvariant that reports an assay result associated with the absence of thedisease or condition. The value of sensitivity, therefore, reflects theprobability that a given diagnostic assay performed on a known variantor diseased sample will produce a result indicative of the presence ofthe variation or disease. As defined here, the clinical relevance of acalculated sensitivity value represents an estimation of the probabilitythat a given assay would detect the presence of a clinical conditionwhen applied to a subject with that condition. Using the technologydescribed herein, it may be possible to achieve a certain level ofaccuracy without the need for generating sequence reads. The accuracymay refer to sensitivity, it may refer to specificity, or it may referto some combination thereof. The desired level of accuracy may bebetween 90% and 95%; it may be between 95% and 98%; it may be between98% and 99%; it may be between 99% and 99.5%; it may be between 99.5%and 99.9%; it may be between 99.9% and 99.99%; it may be between 99.99%and 99.999%, it may be between 99.999% and 100%. Levels of accuracyabove 95% may be referred to as high accuracy.

As used herein, the “specificity” of a given assay (or set of assaysused together) refers to the percentage of normal samples that report anassay result associated with the presence of the disease or condition tobe detected, and a false positive is defined as a clinically-confirmednormal sample that reports an assay result associated with the presenceof the disease or condition. The value of specificity, therefore,reflects the probability that a given diagnostic assay performed on aknown normal sample will produce a result indicative of the presence ofthe variation or disease. As defined here, the clinical relevance of thecalculated specificity value represents an estimation of the probabilitythat a given marker would detect the absence of a clinical conditionwhen applied to a subject without that condition.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The term “genic region” as used herein refers to a gene, its exons, itsintrons, and its regions flanking it upstream and downstream, e.g., 5 to10 kilobases 5′ and 3′ of the transcription start and stop sites,respectively.

The term “genic sequence” as used herein refers to the sequence of agene, its introns, and its regions flanking it upstream and downstream,e.g., 5 to 10 kilobases 5′ and 3′ of the transcription start and stopsites, respectively.

The term “chromosome-specific” as used herein refers to a sequence thatis found only in that particular type of chromosome.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated, e.g., in the presence ofnucleotides and a suitable nucleic acid polymerase. An oligonucleotide“primer” may occur naturally, may be made using molecular biologicalmethods, e.g., purification of a restriction digest, or may be producedsynthetically. In preferred embodiments, a primer is composed of orcomprises DNA.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides including but not limited to analogsthat have altered stacking interactions such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogenbonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner); non-hydrogen bonding analogs (e.g., non-polar, aromaticnucleoside analogs such as 2,4-difluorotoluene, described by B. A.Schweitzer and E. T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B. A.Schweitzer and E. T. Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872);“universal” bases such as 5-nitroindole and 3-nitropyrrole; anduniversal purines and pyrimidines (such as “K” and “P” nucleotides,respectively; P. Kong, et al., Nucleic Acids Res., 1989, 17,10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152).Nucleotide analogs include base analogs, and comprise modified forms ofdeoxyribonucleotides as well as ribonucleotides, and include but are notlimited to modified bases and nucleotides described in U.S. Pat. Nos.5,432,272; 6,001,983; 6,037,120; 6,140,496; 5,912,340; 6,127,121 and6,143,877, each of which is incorporated herein by reference in theirentireties; heterocyclic base analogs based on the purine or pyrimidinering systems, and other heterocyclic bases.

The term “continuous strand of nucleic acid” as used herein is means astrand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

The term “continuous duplex” as used herein refers to a region of doublestranded nucleic acid in which there is no disruption in the progressionof basepairs within the duplex (i.e., the base pairs along the duplexare not distorted to accommodate a gap, bulge or mismatch with theconfines of the region of continuous duplex). As used herein the termrefers only to the arrangement of the basepairs within the duplex,without implication of continuity in the backbone portion of the nucleicacid strand. Duplex nucleic acids with uninterrupted basepairing, butwith nicks in one or both strands are within the definition of acontinuous duplex.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

As applied to polynucleotides, the term “substantial identity” denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence, which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a splice variant of the full-length sequences.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include butare not limited to dyes; radiolabels such as ³²P; binding moieties suchas biotin; haptens such as digoxigenin; luminogenic, phosphorescent orfluorogenic moieties; mass tags; and fluorescent dyes alone or incombination with moieties that can suppress (“quench”) or shift emissionspectra by fluorescence resonance energy transfer (FRET). FRET is adistance-dependent interaction between the electronic excited states oftwo molecules (e.g., two dye molecules, or a dye molecule and anon-fluorescing quencher molecule) in which excitation is transferredfrom a donor molecule to an acceptor molecule without emission of aphoton. (Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995,Methods Enzymol., 246:300, each incorporated herein by reference). Asused herein, the term “donor” refers to a fluorophore that absorbs at afirst wavelength and emits at a second, longer wavelength. The term“acceptor” refers to a moiety such as a fluorophore, chromophore, orquencher that has an absorption spectrum that overlaps the donor'semission spectrum, and that is able to absorb some or most of theemitted energy from the donor when it is near the donor group (typicallybetween 1-100 nm). If the acceptor is a fluorophore, it generally thenre-emits at a third, still longer wavelength; if it is a chromophore orquencher, it then releases the energy absorbed from the donor withoutemitting a photon. In some embodiments, changes in detectable emissionfrom a donor dye (e.g. when an acceptor moiety is near or distant) aredetected. In some embodiments, changes in detectable emission from anacceptor dye are detected. In preferred embodiments, the emissionspectrum of the acceptor dye is distinct from the emission spectrum ofthe donor dye such that emissions from the dyes can be differentiated(e.g., spectrally resolved) from each other.

In some embodiments, a donor dye is used in combination with multipleacceptor moieties. In a preferred embodiment, a donor dye is used incombination with a non-fluorescing quencher and with an acceptor dye,such that when the donor dye is close to the quencher, its excitation istransferred to the quencher rather than the acceptor dye, and when thequencher is removed (e.g., by cleavage of a probe), donor dye excitationis transferred to an acceptor dye. In particularly preferredembodiments, emission from the acceptor dye is detected. See, e.g.,Tyagi, et al., Nature Biotechnology 18:1191 (2000), which isincorporated herein by reference.

Labels may provide signals detectable by fluorescence (e.g., simplefluorescence, FRET, time-resolved fluorescence, fluorescencepolarization, etc.), radioactivity, colorimetry, gravimetry, X-raydiffraction or absorption, magnetism, enzymatic activity,characteristics of mass or behavior affected by mass (e.g., MALDItime-of-flight mass spectrometry), and the like. A label may be acharged moiety (positive or negative charge) or alternatively, may becharge neutral. Labels can include or consist of nucleic acid or proteinsequence, so long as the sequence comprising the label is detectable.

In some embodiment a label comprises a particle for detection. Inpreferred embodiments, the particle is a phosphor particle. Inparticularly preferred embodiments, the phosphor particle is anup-converting phosphor particle (see, e.g., Ostermayer, F. W.Preparation and properties of infrared-to-visible conversion phosphors.Metall. Trans. 752, 747-755 [1971]). In some embodiments, rareearth-doped ceramic particles are used as phosphor particles. Phosphorparticles may be detected by any suitable method, including but notlimited to up-converting phosphor technology (UPT), in whichup-converting phosphors transfer low energy infrared (IR) radiation tohigh-energy visible light. While the present invention is not limited toany particular mechanism, in some embodiments the UPT up-convertsinfrared light to visible light by multi-photon absorption andsubsequent emission of dopant-dependent phosphorescence. See, e.g., U.S.Pat. No. 6,399,397, Issued Jun. 4, 2002 to Zarling, et al.; van DeRijke, et al., Nature Biotechnol. 19(3):273-6 [2001]; Corstjens, et al.,IEE Proc. Nanobiotechnol. 152(2):64 [2005], each incorporated byreference herein in its entirety.

As used herein, the terms “solid support” or “support” refer to anymaterial that provides a substrate structure to which another materialcan be attached. A support or substrate may be, but need not be, solid.Support materials include smooth solid supports (e.g., smooth metal,glass, quartz, plastic, silicon, wafers, carbon (e.g., diamond), andceramic surfaces, etc.), as well as textured and porous materials. Solidsupports need not be flat. Supports include any type of shape, includingspherical shapes (e.g., beads). Support materials also include, but arenot limited to, gels, hydrogels, aerogels, rubbers, polymers, and otherporous and/or non-rigid materials.

As used herein, the terms “bead” and “particle” are usedinterchangeably, and refer to a small support, typically a solidsupport, that is capable of moving about when in a solution (e.g., ithas dimensions smaller than those of the enclosure or container in whichthe solution resides). In some embodiments, beads may settle out of asolution when the solution is not mixed (e.g., by shaking, thermalmixing, vortexing), while in other embodiments, beads may be suspendedin solution in a colloidal fashion. In some embodiments, beads arecompletely or partially spherical or cylindrical. However, beads are notlimited to any particular three-dimensional shape. In some embodiments,beads or particles may be paramagnetic. For example, in someembodiments, beads and particles comprise a magnetic material, e.g.,ferrous oxide.

A bead or particle is not limited to any particular size, and in apreparation comprising a plurality of particles, the particles may beessentially uniform in size (e.g., in diameter) or may be a mixture ofdifferent sizes. In some embodiments, beads comprise or consist ofnanoparticles, e.g., particles of less than about 1000 nm, 900 nm, 800nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, or 1 nm indiameter. In some embodiments, the nanoparticle beads between 5 and 20nm average diameter.

Materials attached to a solid support may be attached to any portion ofthe solid support (e.g., may be attached to an interior portion of aporous solid support material, or to an exterior portion, or to a flatportion on an otherwise non-flat support, or vice versa). In preferredembodiments of the technology, biological molecules such as nucleic acidor protein molecules are attached to solid supports. A biologicalmaterial is “attached” to a solid support when it is affixed to thesolid support through chemical or physical interaction. In someembodiments, attachment is through a covalent bond. However, attachmentsneed not be covalent and need not be permanent. In some embodiments, anattachment may be undone or disassociated by a change in condition,e.g., by temperature, ionic change, addition or removal of a chelatingagent, or other changes in the solution conditions to which the surfaceand bound molecule are exposed.

In some embodiments, materials are attached to a first support and arelocalized to the surface of a second support. For example, in someembodiments, materials that comprise a ferrous or magnetic particle maybe magnetically localized to a surface or a region of a surface, such asa planar surface of a slide or well.

As used herein in reference to a support or substrate, e.g., for acoating or for attachment of a molecule, the term “surface” broadlyrefers to a portion of a support or substrate that is accessible for apurpose. For example, a portion of a bead or vessel or plate that isaccessible to be coated, functionalized, attached to a moiety, e.g., anoligonucleotide or other macromolecule, or otherwise treated, may beconsidered a “surface” of the bead or plate, even if the surface is onan interior portion of the bead or vessel (e.g., within a pore, within asintered matrix, inside a well, etc.) Similarly, a portion of a matrixthat is flexible and/or porous (e.g., a hydrogel, aerogel, mesh, andthat is accessible for a purpose, e.g., to be coated, functionalized,attached to a moiety, derivatized, etc., may be considered a surface ofthe matrix. In certain embodiments, a support may comprise a supportsurface, sometimes termed a first surface, which is the surface of thestructural support material, e.g., in the absence of a coating ormodifying layer, and may further comprise substrate surface, sometimestermed a second surface, which is the surface that is accessible for apurpose after the support surface is modified, e.g., by coating with apolymer or other coating. In some embodiments, the substrate surfacecomprises functional groups capable of complexing covalently ornon-covalently with the one or more analytes, such as oligonucleotidesor polypeptides that comprise reactive or binding groups suitable forcomplexing with the substrate surface functional groups.

As used herein, the term “detergent” refers any of a group of synthetic,organic, liquid or water-soluble agents that have wetting-agent andemulsifying-agent properties, and include anionic agents (e.g., sodiumdodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate,cationic (e.g., benzalkonium chloride, cetyltrimethylammonium bromide)linear alkylbenzene sulfonates (e.g., sodium dodecylbenzene sulfonate),non-ionic (e.g., a TWEEN (e.g., polyoxyethylene (20)sorbitan-monolaurate, -monopalmitate, -monostearate, or -monooleate);TRITON (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether,steroid and steroidal al glycosides (e.g., saponin, digitonin); andzwitterionic (net neutral) agents such as3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),compounds. some embodiments, a “detergent” comprises a mixture ofagents, e.g., TEEPOL® detergent, comprising sodium dodecylbenzenesulfonate, sodium C₁₂-C₁₅ alcohol ether sulfate.

In some embodiments, a target molecule, e.g., a biological material, isattached to a solid support through a “spacer molecule” or “linkergroup.” Such spacer molecules are molecules that have a first portionthat attaches to the biological material and a second portion thatattaches to the solid support. Spacer molecules typically comprise achain of atoms, e.g., carbon atoms, that provide additional distancebetween the first portion and the second portion. Thus, when attached tothe solid support, the spacer molecule permits separation between thesolid support and the biological material, but is attached to both.Examples of linkers and spacers include but are not limited to carbonchains, e.g., C3 and C6 (hexanediol), 1′,2′-dideoxyribose (dSpacer);photocleavable (PC) spacers; triethylene glycol (TEG); and hexa-ethyleneglycol spacers (Integrated DNA Technologies, Inc.).

As used herein, the terms “array” and “microarray” refer a surface orvessel comprising a plurality of pre-defined loci that are addressablefor analysis of the locus, e.g., to determine a result of an assay.Analysis at a locus in an array is not limited to any particular type ofanalysis and includes, e.g., analysis for detection of an atom,molecule, chemical reaction, light or fluorescence emission,suppression, or alteration (e.g., in intensity or wavelength) indicativeof a result at that locus. Examples of pre-defined loci include a gridor any other pattern, wherein the locus to be analyzed is determined byits known position in the array pattern. Microarrays, for example, aredescribed generally in Schena, “Microarray Biochip Technology,” EatonPublishing, Natick, Mass., 2000. Examples of arrays include but are notlimited to supports with a plurality of molecules non-randomly bound tothe surface (e.g., in a grid or other regular pattern) and vesselscomprising a plurality of defined reaction loci (e.g., wells) in whichmolecules or signal-generating reactions may be detected. In someembodiments, an array comprises a patterned distribution of wells thatreceive beads, e.g., as described above for the SIMOA technology. Seealso U.S. Pat. Nos. 9,057,730; 9,556,429; 9,481,883; and 9,376,677, eachof which is incorporated herein by reference in its entirety, for allpurposes.

As used herein, the terms “dispersed” and “dispersal” as used inreference to loci or sites, e.g., on a support or surface, refers to acollection of loci or sites that are distributed or scattered on orabout the surface, wherein at least some of the loci are sufficientlyseparated from other loci that they are individually detectable orresolvable, one from another, e.g., by a detector such as a microscope.Dispersed loci may be in an ordered array, or they may be in anirregular distribution or dispersal, as described below.

As used herein, the term “irregular” as used in reference to a dispersalor distribution of loci or sites, e.g., on a solid support or surface,refers to distribution of loci on or in a surface in a non-arrayedmanner. For example, molecules may be irregularly dispersed on a surfaceby application of a solution of a particular concentration that providesa desired approximate average distance between the molecules on thesurface, but at sites that are not pre-defined by or addressable anypattern on the surface or by the means of applying the solution (e.g.,inkjet printing). In such embodiments, analysis of the surface maycomprise finding the locus of a molecule by detection of a signalwherever it may appear (e.g., scanning a whole surface to detectfluorescence anywhere on the surface). This contrasts to locating asignal by analysis of a surface or vessel only at predetermined loci(e.g., points in a grid array), to determine how much (or what type of)signal appears at each locus in the grid.

As used herein, the term “distinct” in reference to signals refers tosignals that can be differentiated one from another, e.g., by spectralproperties such as fluorescence emission wavelength, color, absorbance,mass, size, fluorescence polarization properties, charge, etc., or bycapability of interaction with another moiety, such as with a chemicalreagent, an enzyme, an antibody, etc.

As used herein, the term “nucleic acid detection assay” refers to anymethod of determining the nucleotide composition of a nucleic acid ofinterest. Nucleic acid detection assay include but are not limited to,DNA sequencing methods, probe hybridization methods, structure specificcleavage assays (e.g., the INVADER assay, (Hologic, Inc.) and aredescribed, e.g., in U.S. Pat. Nos. 5,846,717; 5,985,557; 5,994,069;6,001,567; 6,090,543; and 6,872,816; Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Pat. No.9,096,893, each of which is herein incorporated by reference in itsentirety for all purposes); enzyme mismatch cleavage methods (e.g.,Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, hereinincorporated by reference in their entireties); polymerase chainreaction (PCR), described above; branched hybridization methods (e.g.,Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802,herein incorporated by reference in their entireties); rolling circleamplification (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502,herein incorporated by reference in their entireties); the variation ofrolling circle amplification called “RAM amplification” (see, e.g., U.S.Pat. No. 5,942,391, incorporated herein by reference in its entirety;NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by referencein its entirety); molecular beacon technology (e.g., U.S. Pat. No.6,150,097, herein incorporated by reference in its entirety); E-sensortechnology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170,and 6,063,573, herein incorporated by reference in their entireties);cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and5,660,988, herein incorporated by reference in their entireties); DadeBehring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001,6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated byreference in their entireties); ligase chain reaction (e.g., BaranyProc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridizationmethods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by referencein its entirety).

In some embodiments, target nucleic acid is amplified (e.g., by PCR) andamplified nucleic acid is detected simultaneously using an invasivecleavage assay. Assays configured for performing a detection assay(e.g., invasive cleavage assay) in combination with an amplificationassay are described in U.S. Pat. No. 9,096,893, incorporated herein byreference in its entirety for all purposes. Additional amplificationplus invasive cleavage detection configurations, termed the QuARTSmethod, are described in, e.g., in U.S. Pat. Nos. 8,361,720; 8,715,937;8,916,344; and 9,212,392, each of which is incorporated herein byreference for all purposes. The term “invasive cleavage structure” asused herein refers to a cleavage structure comprising i) a targetnucleic acid, ii) an upstream nucleic acid (e.g., an invasive or“INVADER” oligonucleotide), and iii) a downstream nucleic acid (e.g., aprobe), where the upstream and downstream nucleic acids anneal tocontiguous regions of the target nucleic acid, and where an overlapforms between the a 3′ portion of the upstream nucleic acid and duplexformed between the downstream nucleic acid and the target nucleic acid.An overlap occurs where one or more bases from the upstream anddownstream nucleic acids occupy the same position with respect to atarget nucleic acid base, whether or not the overlapping base(s) of theupstream nucleic acid are complementary with the target nucleic acid,and whether or not those bases are natural bases or non-natural bases.In some embodiments, the 3′ portion of the upstream nucleic acid thatoverlaps with the downstream duplex is a non-base chemical moiety suchas an aromatic ring structure, e.g., as disclosed, for example, in U.S.Pat. No. 6,090,543, incorporated herein by reference in its entirety. Insome embodiments, one or more of the nucleic acids may be attached toeach other, e.g., through a covalent linkage such as nucleic acidstem-loop, or through a non-nucleic acid chemical linkage (e.g., amulti-carbon chain). As used herein, the term “flap endonuclease assay”includes “INVADER” invasive cleavage assays and QuARTS assays, asdescribed above.

As used herein, the terms “digital PCR,” “single molecule PCR” and“single molecule amplification” refer to PCR and other nucleic acidamplification methods that are configured to provide amplificationproduct or signal from a single starting molecule. Typically, samplesare divided, e.g., by serial dilution or by partition into small enoughportions (e.g., in microchambers or in emulsions) such that each portionor dilution has, on average as assessed according to Poissondistribution, no more than a single copy of the target nucleic acid.Methods of single molecule PCR are described, e.g., in U.S. Pat. No.6,143,496, which relates to a method comprising dividing a sample intomultiple chambers such that at least one chamber has at least onetarget, and amplifying the target to determine how many chambers had atarget molecule; U.S. Pat. No. 6,391,559; which relates to an assemblyfor containing and portioning fluid; and U.S. Pat. No. 7,459,315, whichrelates to a method of dividing a sample into an assembly with samplechambers where the samples are partitioned by surface affinity to thechambers, then sealing the chambers with a curable “displacing fluid.”See also U.S. Pat. Nos. 6,440,706 and 6,753,147, and Vogelstein, et al.,Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9236-9241, August 1999. See alsoUS 20080254474, describing a combination of digital PCR combined withmethylation detection.

The term “sequencing”, as used herein, is used in a broad sense and mayrefer to any technique known in the art that allows the order of atleast some consecutive nucleotides in at least part of a nucleic acid tobe identified, including without limitation at least part of anextension product or a vector insert. In some embodiments, sequencingallows the distinguishing of sequence differences between differenttarget sequences. Exemplary sequencing techniques include targetedsequencing, single molecule real-time sequencing, electronmicroscopy-based sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, targeted sequencing, exon sequencing, whole-genomesequencing, sequencing by hybridization, pyrosequencing, capillaryelectrophoresis, gel electrophoresis, duplex sequencing, cyclesequencing, single-base extension sequencing, solid-phase sequencing,high-throughput sequencing, massively parallel signature sequencing,emulsion PCR, co-amplification at lower denaturation temperature-PCR(COLD-PCR), multiplex PCR, sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, ion semiconductor sequencing, nanoballsequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzersequencing, miSeq (Illumina), HiSeq 2000 (Illumina), HiSeq 2500(Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGM™ (LifeTechnologies), MinION™ (Oxford Nanopore Technologies), real-time SMRT™technology (Pacific Biosciences), the Probe-Anchor Ligation (cPAL™)(Complete Genomics/BGI), SOLiD® sequencing, MS-PET sequencing, massspectrometry, and a combination thereof. In some embodiments, sequencingcomprises detecting the sequencing product using an instrument, forexample but not limited to an ABI PRISM® 377 DNA Sequencer, an ABIPRISM® 310, 3100, 3100-Avant, 3730, or 373OxI Genetic Analyzer, an ABIPRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiD™ System (allfrom Applied Biosystems), a Genome Sequencer 20 System (Roche AppliedScience), or a mass spectrometer. In certain embodiments, sequencingcomprises emulsion PCR. In certain embodiments, sequencing comprises ahigh throughput sequencing technique, for example but not limited to,massively parallel signature sequencing (MPSS).

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably in reference to a chain of two or more amino acidslinked together by peptide bonds. Polypeptides may be synthetic ornaturally occurring, and may be short, e.g., between two about 30 aminoacid residues, or may be hundreds or thousands of amino acid residues inlength. Polypeptides may be composed of the 20 main naturally-occurringamino acids, or may comprise one or more non-natural amino acids, e.g.,peptide nucleic acid residues, which comprise pyrimidine or purine baseson a peptide chain backbone, or modified versions of natural amino acids(e.g., modified in the structure of the side groups).

As used herein, the term “antibody” (Ab) refers to antigen-bindingimmunoglobulins, and includes monoclonal antibodies (mAbs) andpolyclonal Abs. The term further includes all modified forms ofantibodies that have the ability to bind to an antigen, e.g., fragmentantibodies (fAbs) comprising portions of an immunoglobulin structure.

As used herein, the term “lectins” refers to a class of non-antibodyproteins that specifically binds to sugars and to sugar moieties (e.g.,sugar moieties on glycoproteins and glucolipids, or within complexcarbohydrates).

As used herein, the terms “crowding agent” and “volume excluder,” asused in reference to a component of a fluid reaction mixture, are usedinterchangeably and refer to compounds, generally polymeric compounds,that reduce available fluid volume in a reaction mixture, therebyincreasing the effective concentration of reactant macromolecules (e.g.,nucleic acids, enzymes, etc.) Crowding reagents include, e.g., glycerol,ethylene glycol, polyethylene glycol, ficoll, serum albumin, casein, anddextran.

As used herein, the terms “digital sequencing,” “single-moleculesequencing,” and “next generation sequencing (NGS)” are usedinterchangeably and refer to determining the nucleotide sequence ofindividual nucleic acid molecules. Systems for individual moleculesequencing include but are not limited to the 454 FLX™ or 454 TITANIUM™(Roche), the SOLEXA™/Illumina Genome Analyzer (Illumina), the HELISCOPE™Single Molecule Sequencer (Helicos Biosciences), and the SOLID™ DNASequencer (Life Technologies/Applied Biosystems) instruments), as wellas other platforms still under development by companies such asIntelligent Biosystems and Pacific Biosystems. See also U.S. Pat. No.7,888,017, entitled “Non-invasive fetal genetic screening by digitalanalysis,” relating to digital analysis of maternal and fetal DNA, e.g.,cfDNA.

As used herein, the term “probe” or “hybridization probe” refers to anoligonucleotide (i.e., a sequence of nucleotides), whether occurringnaturally as in a purified restriction digest or produced synthetically,recombinantly or by PCR amplification, that is capable of hybridizing,at least in part, to another oligonucleotide of interest. A probe may besingle-stranded or double-stranded. Probes are useful in the detection,identification and isolation of particular sequences. In some preferredembodiments, probes used in the present invention will be labeled with a“reporter molecule,” so that is detectable in any detection system,including, but not limited to enzyme (e.g., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. It is not intended that the present invention belimited to any particular detection system or label.

The term “MIP” as used herein, refers to a molecular inversion probe (ora circular capture probe). Molecular inversion probes (or circularcapture probes) are nucleic acid molecules that comprise a pair ofunique polynucleotide arms that hybridize to a target nucleic acid toform a nick or gap and a polynucleotide linker (e.g., a universalbackbone linker). In some embodiments, the unique polynucleotide armshybridize to a target strand immediately adjacent to each other to forma ligatable nick (generally termed “padlock probes”) while in someembodiments, one the hybridized MIP must be further modified (e.g., bypolymerase extension, base excision, and/or flap cleavage) to form aligatable nick. Ligation of a MIP probe to form a circular nucleic acidis typically indicative of the presence of the complementary targetstrand. In some embodiments, MIPs comprise one or more unique moleculartags (or unique molecular identifiers). See, for example, FIG. 1. Insome embodiments, a MIP may comprise more than one unique moleculartags, such as, two unique molecular tags, three unique molecular tags,or more. In some embodiments, the unique polynucleotide arms in each MIPare located at the 5′ and 3′ ends of the MIP, while the unique moleculartag(s) and the polynucleotide linker are located internal to the 5′ and3′ ends of the MIP. For example, the MIPs that are used in someembodiments of this disclosure comprise in sequence the followingcomponents: first unique polynucleotide arm—first unique moleculartag—polynucleotide linker—second unique molecular tag—second uniquepolynucleotide arm. In some embodiments, the MIP is a 5′ phosphorylatedsingle-stranded nucleic acid (e.g., DNA) molecule. See, for example, WO2017/020023, filed Jul. 29, 2016, and WO 2017/020024, filed Jul. 29,2016, each of which is incorporated by reference herein for allpurposes.

As used herein, the terms “circular nucleic acid” and “circularizednucleic acid” as used, for example, in reference to probe nucleic acids,refers to nucleic acid strands that are joined at the ends, e.g., byligation, to form a continuous circular strand of nucleic acid.

The unique molecular tag may be any tag that is detectable and can beincorporated into or attached to a nucleic acid (e.g., a polynucleotide)and allows detection and/or identification of nucleic acids thatcomprise the tag. In some embodiments the tag is incorporated into orattached to a nucleic acid during sequencing (e.g., by a polymerase).Non-limiting examples of tags include nucleic acid tags, nucleic acidindexes or barcodes, radiolabels (e.g., isotopes), metallic labels,fluorescent labels, chemiluminescent labels, phosphorescent labels,fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies orparts thereof, linkers, members of a binding pair), the like orcombinations thereof. In some embodiments, particularly sequencingembodiments, the tag (e.g., a molecular tag) is a unique, known and/oridentifiable sequence of nucleotides or nucleotide analogues (e.g.,nucleotides comprising a nucleic acid analogue, a sugar and one to threephosphate groups). In some embodiments, tags are six or more contiguousnucleotides. A multitude of fluorophore-based tags are available with avariety of different excitation and emission spectra. Any suitable typeand/or number of fluorophores can be used as a tag. In some embodiments1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 ormore, 100 or more, 500 or more, 1000 or more, 10,000 or more, 100,000 ormore different tags are utilized in a method described herein (e.g., anucleic acid detection and/or sequencing method). In some embodiments,one or two types of tags (e.g., different fluorescent labels) are linkedto each nucleic acid in a library. In some embodiments,chromosome-specific tags are used to make chromosomal counting faster ormore efficient. Detection and/or quantification of a tag can beperformed by a suitable method, machine or apparatus, non-limitingexamples of which include flow cytometry, quantitative polymerase chainreaction (qPCR), gel electrophoresis, a luminometer, a fluorometer, aspectrophotometer, a suitable gene-chip or microarray analysis, Westernblot, mass spectrometry, chromatography, cytofluorimetric analysis,fluorescence microscopy, a suitable fluorescence or digital imagingmethod, confocal laser scanning microscopy, laser scanning cytometry,affinity chromatography, manual batch mode separation, electric fieldsuspension, a suitable nucleic acid sequencing method and/or nucleicacid sequencing apparatus, the like and combinations thereof.

In the MIPs, the unique polynucleotide arms are designed to hybridizeimmediately upstream and downstream of a specific target sequence (orsite) in a nucleic acid target, e.g., in an RNA, cfDNA, or genomicnucleic acid sample. In some embodiments, hybridization of a MIP to atarget sequence produces a ligatable nick without a gap, i.e., the twoarms of the MIP hybridize to contiguous sequences in the target strandsuch that no overlap or gap is formed upon hybridization. Such zero-gapMIPs are generally termed “padlock” probes. See, e.g., M. Nilsson, etal. “Padlock probes: circularizing oligonucleotides for localized DNAdetection”. Science. 265 (5181): 2085-2088 (1994); J. Banér, et al.,Nucleic Acids Res., 26 (22):5073-5078 (1998). In other embodiments thehybridized MIP/target nucleic acid complex requires modification toproduce a ligatable nick. For example, in some embodiments,hybridization leaves a gap that is filled, e.g., by polymerase extendinga 3′ end of the MIP, prior to ligation, while in other embodiments,hybridization forms an overlapping flap structure that must be modified,e.g., by a flap endonuclease or a 3′ exonuclease, to produce a ligatablenick. In some embodiments, MIPS comprise unique molecular tags are shortnucleotide sequences that are randomly generated. In some embodiments,the unique molecular tags do not hybridize to any sequence or sitelocated on a genomic nucleic acid fragment or in a genomic nucleic acidsample. In some embodiments, the polynucleotide linker (or the backbonelinker) in the MIPs are universal in all the MIPs used in embodiments ofthis disclosure.

In some embodiments, the MIPs are introduced to nucleic acid fragmentsderived from a test subject (or a reference subject) to perform captureof target sequences or sites (or control sequences or sites) located ona nucleic acid sample (e.g., a genomic DNA). In some embodiments,fragmenting aids in capture of target nucleic acid by molecularinversion probes. In some embodiments, for example, when the nucleicacid sample is comprised of cell free nucleic acid, fragmenting may notbe necessary to improve capture of target nucleic acid by molecularinversion probes. For example, in some types of samples, cell freenucleic acid is fragmented in the sample such that further fragmentationis not necessary and may even be detrimental capture of the targetnucleic acids. As described in greater detail herein, after capture ofthe target sequence (e.g., locus) of interest, the captured target maybe subjected to enzymatic gap-filling and ligation steps, such that acopy of the target sequence is incorporated into a circle-likestructure. In some embodiments, nucleic acid analogs, e.g., containinglabels, haptens, etc., may be incorporated in the filled section, foruse, e.g., in downstream detection, purification, or other processingsteps. Capture efficiency of the MIP to the target sequence on thenucleic acid fragment can, in some embodiments, be improved bylengthening the hybridization and gap-filling incubation periods. (See,e.g., Turner E H, et al., Nat Methods. 2009 Apr. 6:1-2.).

In some embodiments, the MIPs that are used according to the disclosureto capture a target site or target sequence comprise in sequence thefollowing components: first targeting polynucleotide arm—first uniquetargeting molecular tag—polynucleotide linker—second unique targetingmolecular tag—second targeting polynucleotide arm.

In some embodiments, the MIPs that are used in the disclosure to capturea control site or control sequence comprise in sequence the followingcomponents: first control polynucleotide arm—first unique controlmolecular tag—polynucleotide linker—second unique control moleculartag—second control polynucleotide arm.

MIP technology may be used to detect or amplify particular nucleic acidsequences in complex mixtures. One of the advantages of using the MIPtechnology is in its capacity for a high degree of multiplexing, whichallows thousands of target sequences to be captured in a single reactioncontaining thousands of MIPs. Various aspects of MIP technology aredescribed in, for example, Hardenbol et al., “Multiplexed genotypingwith sequence-tagged molecular inversion probes,” Nature Biotechnology,21(6): 673-678 (2003); Hardenbol et al., “Highly multiplexed molecularinversion probe genotyping: Over 10,000 targeted SNPs genotyped in asingle tube assay,” Genome Research, 15: 269-275 (2005); Burmester etal., “DMET microarray technology for pharmacogenomics-based personalizedmedicine,” Methods in Molecular Biology, 632: 99-124 (2010); Sissung etal., “Clinical pharmacology and pharmacogenetics in a genomics era: theDMET platform,” Pharmacogenomics, 11(1): 89-103 (2010); Deeken, “TheAffymetrix DMET platform and pharmacogenetics in drug development,”Current Opinion in Molecular Therapeutics, 11(3): 260-268 (2009); Wanget al., “High quality copy number and genotype data from FFPE samplesusing Molecular Inversion Probe (MIP) microarrays,” BMC MedicalGenomics, 2:8 (2009); Wang et al., “Analysis of molecular inversionprobe performance for allele copy number determination,” Genome Biology,8(11): R246 (2007); Ji et al., “Molecular inversion probe analysis ofgene copy alternations reveals distinct categories of colorectalcarcinoma,” Cancer Research, 66(16): 7910-7919 (2006); and Wang et al.,“Allele quantification using molecular inversion probes (MIP),” NucleicAcids Research, 33(21): e183 (2005), each of which is herebyincorporated by reference in its entirety for all purposes. See also inU.S. Pat. Nos. 6,858,412; 5,817,921; 6,558,928; 7,320,860; 7,351,528;5,866,337; 6,027,889 and 6,852,487, each of which is hereby incorporatedby reference in its entirety for all purposes.

MIP technology has previously been successfully applied to other areasof research, including the novel identification and subclassification ofbiomarkers in cancers. See, e.g., Brewster et al., “Copy numberimbalances between screen- and symptom-detected breast cancers andimpact on disease-free survival,” Cancer Prevention Research, 4(10):1609-1616 (2011); Geiersbach et al., “Unknown partner for USP6 andunusual SS18 rearrangement detected by fluorescence in situhybridization in a solid aneurysmal bone cyst,” Cancer Genetics, 204(4):195-202 (2011); Schiffman et al., “Oncogenic BRAF mutation with CDKN2Ainactivation is characteristic of a subset of pediatric malignantastrocytomas,” Cancer Research, 70(2): 512-519 (2010); Schiffman et al.,“Molecular inversion probes reveal patterns of 9p21 deletion and copynumber aberrations in childhood leukemia,” Cancer Genetics andCytogenetics, 193(1): 9-18 (2009); Press et al., “Ovarian carcinomaswith genetic and epigenetic BRCA1 loss have distinct molecularabnormalities,” BMC Cancer, 8:17 (2008); and Deeken et al., “Apharmacogenetic study of docetaxel and thalidomide in patients withcastration-resistant prostate cancer using the DMET genotypingplatform,” Pharmacogenomics, 10(3): 191-199 (2009), each of which ishereby incorporated by reference in its entirety for all purposes.

MIP technology has also been applied to the identification of newdrug-related biomarkers. See, e.g., Caldwell et al., “CYP4F2 geneticvariant alters required warfarin dose,” Blood, 111(8): 4106-4112 (2008);and McDonald et al., “CYP4F2 Is a Vitamin K1 Oxidase: An Explanation forAltered Warfarin Dose in Carriers of the V433M Variant,” MolecularPharmacology, 75: 1337-1346 (2009), each of which is hereby incorporatedby reference in its entirety for all purposes. Other MIP applicationsinclude drug development and safety research. See, e.g., Mega et al.,“Cytochrome P-450 Polymorphisms and Response to Clopidogrel,” NewEngland Journal of Medicine, 360(4): 354-362 (2009); Dumaual et al.,“Comprehensive assessment of metabolic enzyme and transporter genesusing the Affymetrix Targeted Genotyping System,” Pharmacogenomics,8(3): 293-305 (2007); and Daly et al., “Multiplex assay forcomprehensive genotyping of genes involved in drug metabolism,excretion, and transport,” Clinical Chemistry, 53(7): 1222-1230 (2007),each of which is hereby incorporated by reference in its entirety forall purposes. Further applications of MIP technology include genotypeand phenotype databasing. See, e.g., Man et al., “Genetic Variation inMetabolizing Enzyme and Transporter Genes: Comprehensive Assessment in 3Major East Asian Subpopulations with Comparison to Caucasians andAfricans,” Journal of Clinical Pharmacology, 50(8): 929-940 (2010),which is hereby incorporated by reference in its entirety for allpurposes.

The term “capture” or “capturing”, as used herein, refers to the bindingor hybridization reaction between a molecular inversion probe and itscorresponding targeting site. In some embodiments, upon capturing, acircular replicon or a MIP replicon is produced or formed. In someembodiments, the targeting site is a deletion (e.g., partial or fulldeletion of one or more exons). In some embodiments, a target MIP isdesigned to bind to or hybridize with a naturally-occurring (e.g.,wild-type) genomic region of interest where a target deletion isexpected to be located. The target MIP is designed to not bind to agenomic region exhibiting the deletion. In these embodiments, binding orhybridization between a target MIP and the target site of deletion isexpected to not occur. The absence of such binding or hybridizationindicates the presence of the target deletion. In these embodiments, thephrase “capturing a target site” or the phrase “capturing a targetsequence” refers to detection of a target deletion by detecting theabsence of such binding or hybridization. As used in reference to otheroligonucleotides, e.g., “capture oligonucleotide” the term refers to abinding or hybridization reaction between the capture oligonucleotideand a nucleic acid to be captured, e.g., to be immobilized, removed fromsolution, or otherwise be manipulated by hybridization to the captureoligonucleotide.

The term “MIP replicon” or “circular replicon”, as used herein, refersto a circular nucleic acid molecule generated via a capturing reaction(e.g., a binding or hybridization reaction between a MIP and itstargeted sequence). In some embodiments, the MIP replicon is asingle-stranded circular nucleic acid molecule. In some embodiments, atargeting MIP captures or hybridizes to a target sequence or site. Afterthe capturing reaction or hybridization, in some embodiments, a ligationreaction mixture is introduced to ligate the nick formed byhybridization of the two targeting polynucleotide arms to formsingle-stranded circular nucleotide molecules, i.e., a targeting MIPreplicon, while in some embodiments, hybridization of the MIP leaves agap, and a ligation/extension mixture is introduced to extend and ligatethe gap region between the two targeting polynucleotide arms to form atargeting MIP replicon. In some embodiments, a control MIP captures orhybridizes to a control sequence or site. After the capturing reactionor hybridization, a ligation reaction mixture is introduced to ligatethe nick formed by hybridization of the two control polynucleotide arms,or a ligation/extension mixture is introduced to extend and ligate thegap region between the two control polynucleotide arms to formsingle-stranded circular nucleotide molecules, i.e., a control MIPreplicon. MIP replicons may be amplified through a polymerase chainreaction (PCR) to produce a plurality of targeting MIP amplicons, whichare double-stranded nucleic acid molecules. MIP replicons findparticular application in rolling circle amplification, or RCA. RCA isan isothermal nucleic acid amplification technique where a DNApolymerase continuously adds single nucleotides to a primer annealed toa circular template, which results in a long concatemer of singlestranded DNA that contains tens to hundreds to thousands of tandemrepeats (complementary to the circular template). See, e.g., M. Ali, etal. “Rolling circle amplification: a versatile tool for chemicalbiology, materials science and medicine”. Chemical Society Reviews. 43(10): 3324-3341, which is incorporated herein by reference in itsentirety, for all purposes. See also WO 2015/083002, which isincorporated herein by reference in its entirety, for all purposes.

Polymerases typically used in RCA for DNA amplification are Phi29, Bst,and Vent exo-DNA polymerases, with Phi29 DNA polymerase being preferredin view of its superior processivity and strand displacement ability

The term “amplicon”, as used herein, refers to a nucleic acid generatedvia amplification reaction (e.g., a PCR reaction). In some embodiments,the amplicon is a single-stranded nucleic acid molecule. In someembodiments, the amplicon is a double-stranded nucleic acid molecule. Insome embodiments, a targeting MIP replicon is amplified usingconventional techniques to produce a plurality of targeting MIPamplicons, which are double-stranded nucleotide molecules. In someembodiments, a control MIP replicon is amplified using conventionaltechniques to produce a plurality of control MIP amplicons, which aredouble-stranded nucleotide molecules.

The term “probe oligonucleotide” or “flap oligonucleotide” when used inreference to a flap assay (e.g., an INVADER invasive cleavage assay),refers to an oligonucleotide that interacts with a target nucleic acidto form a cleavage structure in the presence of an invasiveoligonucleotide.

The term “invasive oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid at a location adjacent to the regionof hybridization between a probe and the target nucleic acid, whereinthe 3′ end of the invasive oligonucleotide comprises a portion (e.g., achemical moiety, or one or more nucleotides) that overlaps with theregion of hybridization between the probe and target. The 3′ terminalnucleotide of the invasive oligonucleotide may or may not base pair anucleotide in the target. In some embodiments, the invasiveoligonucleotide contains sequences at its 3′ end that are substantiallythe same as sequences located at the 5′ end of a portion of the probeoligonucleotide that anneals to the target strand.

The term “flap endonuclease” or “FEN,” as used herein, refers to a classof nucleolytic enzymes, typically 5′ nucleases, that act asstructure-specific endonucleases on DNA structures with a duplexcontaining a single stranded 5′ overhang, or flap, on one of the strandsthat is displaced by another strand of nucleic acid (e.g., such thatthere are overlapping nucleotides at the junction between the single anddouble-stranded DNA). FENs catalyze hydrolytic cleavage of thephosphodiester bond at the junction of single and double stranded DNA,releasing the overhang, or the flap. Flap endonucleases are reviewed byCeska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al(Annu. Rev. Biochem. 2004 73: 589-615; herein incorporated by referencein its entirety). FENs may be individual enzymes, multi-subunit enzymes,or may exist as an activity of another enzyme or protein complex (e.g.,a DNA polymerase).

A flap endonuclease may be thermostable. For example, FEN-1 flapendonuclease from archival thermophiles organisms are typicalthermostable. As used herein, the term “FEN-1” refers to anon-polymerase flap endonuclease from a eukaryote or archaeal organism.See, e.g., WO 02/070755, and Kaiser M. W., et al. (1999) J. Biol. Chem.,274:21387, which are incorporated by reference herein in theirentireties for all purposes.

As used herein, the term “cleaved flap” refers to a single-strandedoligonucleotide that is a cleavage product of a flap assay.

The term “cassette,” when used in reference to a flap cleavage reaction,refers to an oligonucleotide or combination of oligonucleotidesconfigured to generate a detectable signal in response to cleavage of aflap or probe oligonucleotide, e.g., in a primary or first cleavagestructure formed in a flap cleavage assay. In preferred embodiments, thecassette hybridizes to a non-target cleavage product produced bycleavage of a flap oligonucleotide to form a second overlapping cleavagestructure, such that the cassette can then be cleaved by the sameenzyme, e.g., a FEN-1 endonuclease.

In some embodiments, the cassette is a single oligonucleotide comprisinga hairpin portion (i.e., a region wherein one portion of the cassetteoligonucleotide hybridizes to a second portion of the sameoligonucleotide under reaction conditions, to form a duplex). In otherembodiments, a cassette comprises at least two oligonucleotidescomprising complementary portions that can form a duplex under reactionconditions. In preferred embodiments, the cassette comprises a label,e.g., a fluorophore. In particularly preferred embodiments, a cassettecomprises labeled moieties that produce a FRET effect. In suchembodiments, the cassette may be referred to as a “FRET cassette.” See,for example, U.S. Pat. No. 9,096,893, issued Aug. 4, 2015, which isincorporated herein by reference in its entirety, for all purposes.

As used herein, the phrase “not substantially complementary” as used inreference to a probe flap or arm means that the flap portion issufficiently non-complementary not to hybridize selectively to a nucleicacid sequence, e.g., a target nucleic acid or amplified DNA, under thedesignated annealing conditions or stringent conditions, encompassingthe terms “substantially non-complementary” and “perfectlynon-complementary.”

The term “signal” as used herein refers to any detectable effect, suchas would be caused or provided by a label or by action or accumulationof a component or product in an assay reaction.

As used herein, the term “detector” refers to a system or component of asystem, e.g., an instrument (e.g. a camera, fluorimeter, charge-coupleddevice, scintillation counter, solid state nanopore device, etc.) or areactive medium (X-ray or camera film, pH indicator, etc.), that canconvey to a user or to another component of a system (e.g., a computeror controller) the presence of a signal or effect. A detector is notlimited to a particular type of signal detected, and can be aphotometric or spectrophotometric system, which can detect ultraviolet,visible or infrared light, including fluorescence or chemiluminescence;a radiation detection system; a charge detection system; a system fordetection of an electronic signal, e.g., a current or chargeperturbation; a spectroscopic system such as nuclear magnetic resonancespectroscopy, mass spectrometry or surface enhanced Raman spectrometry;a system such as gel or capillary electrophoresis or gel exclusionchromatography; or other detection system known in the art, orcombinations thereof.

The term “detection” as used herein refers to quantitatively orqualitatively identifying an analyte (e.g., DNA, RNA or a protein),e.g., within a sample. The term “detection assay” as used herein refersto a kit, test, or procedure performed for the purpose of detecting ananalyte within a sample. Detection assays produce a detectable signal oreffect when performed in the presence of the target analyte, and includebut are not limited to assays incorporating the processes ofhybridization, nucleic acid cleavage (e.g., exo- or endonuclease),nucleic acid amplification, nucleotide sequencing, primer extension,nucleic acid ligation, antigen-antibody binding, interaction of aprimary antibody with a secondary antibody, and/or conformational changein a nucleic acid (e.g., an oligonucleotide) or polypeptide (e.g., aprotein or small peptide).

As used herein, the term “prenatal or pregnancy-related disease orcondition” refers to any disease, disorder, or condition affecting apregnant woman, embryo, or fetus. Prenatal or pregnancy-relatedconditions can also refer to any disease, disorder, or condition that isassociated with or arises, either directly or indirectly, as a result ofpregnancy. These diseases or conditions can include any and all birthdefects, congenital conditions, or hereditary diseases or conditions.Examples of prenatal or pregnancy-related diseases include, but are notlimited to, Rhesus disease, hemolytic disease of the newborn,beta-thalassemia, sex determination, determination of pregnancy, ahereditary Mendelian genetic disorder, chromosomal aberrations, a fetalchromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomalmonosomy, trisomy 8, trisomy 13 (Patau Syndrome), trisomy 16, trisomy 18(Edwards syndrome), trisomy 21 (Down syndrome), X-chromosome linkeddisorders, trisomy X (XXX syndrome), monosomy X (Turner syndrome), XXYsyndrome, XYY syndrome, XYY syndrome, XXXY syndrome, XXYY syndrome, XYYYsyndrome, XXXXX syndrome, XXXXY syndrome, XXXYY syndrome, XXYYYsyndrome, Fragile X Syndrome, fetal growth restriction, cystic fibrosis,a hemoglobinopathy, fetal death, fetal alcohol syndrome, sickle cellanemia, hemophilia, Klinefelter syndrome, dup(17)(p11.2p1.2) syndrome,endometriosis, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2)syndrome, cat eye syndrome, cri-du-chat syndrome, Wolf-Hirschhornsyndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease,neuropathy with liability to pressure palsies, Smith-Magenis syndrome,neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, steroid sulfatase deficiency, Prader-Willi syndrome,Kallmann syndrome, microphthalmia with linear skin defects, adrenalhypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease,testis-determining factor on Y, azospermia (factor a), azospermia(factor b), azospermia (factor c), 1p36 deletion, phenylketonuria,Tay-Sachs disease, adrenal hyperplasia, Fanconi anemia, spinal muscularatrophy, Duchenne's muscular dystrophy, Huntington's disease, myotonicdystrophy, Robertsonian translocation, Angelman syndrome, tuberoussclerosis, ataxia telangieltasia, open spina bifida, neural tubedefects, ventral wall defects, small-for-gestational-age, congenitalcytomegalovirus, achondroplasia, Marfan's syndrome, congenitalhypothyroidism, congenital toxoplasmosis, biotinidase deficiency,galactosemia, maple syrup urine disease, homocystinuria, medium-chainacyl Co-A dehydrogenase deficiency, structural birth defects, heartdefects, abnormal limbs, club foot, anencephaly,arhinencephaly/holoprosencephaly, hydrocephaly,anophthalmos/microphthalmos, anotia/microtia, transposition of greatvessels, tetralogy of Fallot, hypoplastic left heart syndrome,coarctation of aorta, cleft palate without cleft lip, cleft lip with orwithout cleft palate, oesophageal atresia/stenosis with or withoutfistula, small intestine atresia/stenosis, anorectal atresia/stenosis,hypospadias, indeterminate sex, renal agenesis, cystic kidney, preaxialpolydactyly, limb reduction defects, diaphragmatic hernia, blindness,cataracts, visual problems, hearing loss, deafness, X-linkedadrenoleukodystrophy, Rett syndrome, lysosomal disorders, cerebralpalsy, autism, aglossia, albinism, ocular albinism, oculocutaneousalbinism, gestational diabetes, Arnold-Chiari malformation, CHARGEsyndrome, congenital diaphragmatic hernia, brachydactlia, aniridia,cleft foot and hand, heterochromia, Dwarnian ear, Ehlers Danlossyndrome, epidermolysis bullosa, Gorham's disease, Hashimoto's syndrome,hydrops fetalis, hypotonia, Klippel-Feil syndrome, muscular dystrophy,osteogenesis imperfecta, progeria, Smith Lemli Opitz syndrome,chromatelopsia, X-linked lymphoproliferative disease, omphalocele,gastroschisis, pre-eclampsia, eclampsia, pre-term labor, prematurebirth, miscarriage, delayed intrauterine growth, ectopic pregnancy,hyperemesis gravidarum, morning sickness, or likelihood for successfulinduction of labor.

In some NIPT embodiments, the technology described herein furtherincludes estimating a fetal fraction for a sample, wherein the fetalfraction is used to aid in the determination of whether the genetic datafrom the test subject is indicative of an aneuploidy. Methods fordetermining or calculating fetal fraction are known in the art.

As used herein, the term “valid detection assay” refers to a detectionassay that has been shown to accurately predict an association betweenthe detection of a target and a phenotype (e.g. medical condition).Examples of valid detection assays include, but are not limited to,detection assays that, when a target is detected, accurately predict thephenotype medical 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of thetime. Other examples of valid detection assays include, but are notlimited to, detection assays that qualify as and/or are marketed asAnalyte-Specific Reagents (i.e. as defined by FDA regulations) orIn-Vitro Diagnostics (i.e. approved by the FDA).

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to a delivery systemcomprising two or more separate containers that each contain asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

As used herein, the term “information” refers to any collection of factsor data. In reference to information stored or processed using acomputer system(s), including but not limited to internets, the termrefers to any data stored in any format (e.g., analog, digital, optical,etc.). As used herein, the term “information related to a subject”refers to facts or data pertaining to a subject (e.g., a human, plant,or animal). The term “genomic information” refers to informationpertaining to a genome including, but not limited to, nucleic acidsequences, genes, allele frequencies, RNA expression levels, proteinexpression, phenotypes correlating to genotypes, etc. “Allele frequencyinformation” refers to facts or data pertaining allele frequencies,including, but not limited to, allele identities, statisticalcorrelations between the presence of an allele and a characteristic of asubject (e.g., a human subject), the presence or absence of an allele inan individual or population, the percentage likelihood of an allelebeing present in an individual having one or more particularcharacteristics, etc.

As used herein, the term “assay validation information” refers togenomic information and/or allele frequency information resulting fromprocessing of test result data (e.g. processing with the aid of acomputer). Assay validation information may be used, for example, toidentify a particular candidate detection assay as a valid detectionassay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram of a molecular inversion probe (MIP)for chromosome-specific recognition, suitable for use in massivelymultiplexed capture assays.

FIG. 2 provides a schematic diagram of an embodiment of multiplexedchromosome-specific rolling circle amplification.

FIG. 3 provides a schematic diagram of an embodiment of multiplexedchromosome-specific rolling circle amplification using molecular beaconprobes for detection.

FIG. 4 provides a schematic diagram of an embodiment of the technologycomprising circularizing cfDNA using a single-strand ligase (e.g.,CircLigase™ thermostable RNA ligase) to make “native circles” fordetection.

FIG. 5 provides a schematic diagram of an embodiment of the technologycomprising circularizing cfDNA and using “Golden Gate Assembly” to addsegments for detection (see, e.g., Engler, C., Kandzia, R., andMarillonnet, S. (2008) PLoS ONE 3, e3647.)

FIG. 6 provides a schematic diagram of an embodiment of the technologycomprising circularizing cfDNA using extension ligation on a uniquemolecular inversion-inducing template, with detection using anembodiment of RCA.

FIG. 7 provides a schematic diagram of an embodiment of the technologycomprising a unique molecular inversion inducing template that isextended and ligated to create a circular DNA molecule, with detectionusing an embodiment of RCA.

FIG. 8 provides a schematic diagram of an embodiment of the technologycomprising a synthetic circular DNA comprising binding sites for probebinding and a primer binding site for replication, for use, e.g., as atemplate for rolling circle amplification.

FIG. 9 provides a schematic diagram of an embodiment of the technologycomprising use of pairs of probes configured for collisional quenchingwhen hybridized to a strand of DNA, for use in detection of product fromRCA.

FIG. 10 provides a schematic diagram of an embodiment of the technologycomprising use of pairs of probes configured for fluorescence resonanceenergy transfer (FRET) when hybridized to a strand of DNA, for use indetection of product from RCA.

FIG. 11 provides a schematic diagram of an embodiment of the technologycomprising use of probes comprising a dye and a quencher, configured tobe cleaved, e.g., using a duplex-specific nuclease, such as arestriction enzyme, when hybridized to a strand of DNA, for use indetection of product from RCA.

FIG. 12 provides a schematic diagram of an embodiment of the technologycomprising use of RCA of CIDS, followed by CID-specific digestion andCID-specific labeling.

FIG. 13 illustrates an embodiment in which MIPs hybridize to targetnucleic acid, e.g., cfDNA, leaving a single nucleotide gap. The gap isfilled by extension to incorporate a biotinylated nucleotide and closedby ligation. The circularized MIPs may then be bound to astreptavidin-coated surface.

FIG. 14 shows a schematic diagram of an initiator oligonucleotidehybridized to a MIP immobilized on a surface.

FIG. 15 shows a schematic diagram of hairpin oligonucleotides that worktogether to form a self-assembling scaffold in the presence of aninitiator oligonucleotide.

FIG. 16 illustrates a self-assembled scaffold comprising multiplelabels, e.g., fluorescent dyes.

FIG. 17 provide a schematic diagram of an invasive cleavage structureaccording to an embodiment of the technology.

FIG. 18 provides an illustration of a hairpin probe for use in formingan invasive cleavage structure for a flap endonuclease assay, e.g., anInvader® assay, according to an embodiment of the technology.

FIG. 19 provides an illustration of the accumulation of cleaved flapfragments in a flap endonuclease assay.

FIG. 20 illustrates an embodiment in which a cleaved biotinylated flapis captured using an immobilized complementary probe, and the biotin isreacted with streptavidin linked to an enzyme, e.g., β-galactosidase.

FIG. 21 illustrates an embodiment of the technology in which MIPsdesigned to target different chromosomes each require a differentnucleotide to extend and ligate, and wherein the MIPs are extended andligated in a chromosome-specific manner using nucleotides which carrydifferent dyes or haptens for each different dNTP.

FIG. 22, panels A, B, and C, illustrate embodiments of the technology inwhich MIPs contain or are modified to contain an immobilization moiety,or are hybridized to an oligonucleotide containing an immobilizationmoiety, and are immobilized on a surface.

FIG. 23 provides a schematic diagram of a rolling circle amplificationreaction.

FIGS. 24A-24D provide graphs showing results from examining the effecton RCA signal of including biotin residues in the MIP complex.

FIGS. 25A-25C provide graphs showing the results of varying amounts ofcomponents in standard RCA reactions in solution.

FIG. 26 provides graphs comparing the effects on signal accumulationfrom signal amplification of single molecules by rolling circleamplification (RCA) of using different molecular weights of PEG at thepercentages (w:v) shown.

FIGS. 27A-27B show results achieved in RCA reactions performed usingprimers bound to glass surfaces in an irregular dispersion, withdetection using molecular beacon probes comprising a quencher andfluorophore.

FIG. 27A shows microscope images of surfaces of APTES-silanized plates,as described in Example 1, and compares RCA signal with or without PEG.

FIG. 27B provides graphs showing the effects of PEG on the numberfluorescent spots and on the size of the spots in pixels for the spotsshown in FIG. 27A.

FIG. 28 provides graphs showing the effects of different molecularweights of PEG in a 20% solution on the number and pixel size of thespots on APTES-silanized plates, as described in Example 1.

FIG. 29A shows microscope images of surfaces of APTES-silanized plates,as described in Example 1, and compares RCA signal for reactionshybridized for 18 hours or 1 hour prior to initiating the RCA reaction.

FIG. 29B provides graphs comparing the effects of hybridization time andbuffer on the number and pixel size (area) of the spots shown in FIG.29A.

FIG. 30 provides graphs comparing the effects of PEG 200 on the standardRCA reaction conditions, with or without a 2-hour hybridization time,and the effect of PEG 2000 with 2-hour hybridization, on the number andpixel size (area) of the spots.

FIG. 31 provides graphs comparing the effects of PEG 200 on the standardRCA reaction conditions performed at 25° C., with or without a 2-hourhybridization time, and the effect of PEG 2000 with 2-hourhybridization, on the number and pixel size (area) of the spots.

FIG. 32 provides graphs comparing the effects of PEG 200 on the standardRCA reaction conditions performed at 37° C., with or without a 2-hourhybridization time, and the effect of PEG 2000 with 2-hourhybridization, on the number and pixel size (area) of the spots.

FIG. 33 shows microscope images of surfaces of APTES-silanized plates,as described in Example 1, and compares RCA signal for reactionscomprising PEG 600 at the indicated concentrations, performed at 37° C.or 45° C.

FIG. 34 provides a schematic diagram of RCA-molecular beacon products ona surface, with or without graphene oxide, with graphene oxide quenchingfluorescence background from beacons bound non-specifically to thesurface.

FIG. 35 provides a schematic diagram of a two-step RCA reaction, inwhich the rolling circle reaction is started, the molecular beacon andgraphene oxide are added, and the RCA reaction is further incubated, asdescribed in Example 1.

FIG. 36 shows microscope images of surfaces of APTES-silanized plates,as described in Example 1, and shows RCA signal for two-step reactionsgraphene oxide.

FIG. 37 provides a graph comparing spot counts for RCA reactions doneone step (no GO) or two steps (with or without GO), comparing reactionswith 100 fmol of circularized MIP to reactions with no circularized MIP.

FIG. 38 provides schematic diagrams of different capture complexes forapplications of embodiments of the technology to detection of differenttypes of target molecules.

FIG. 39 provides a schematic diagram of applications of the technologyto detection of immobilized antigens.

FIG. 40 provides a schematic diagram of applications of the technologyto detection of immobilized antigen-antibody complexes.

FIG. 41 provides a schematic diagram illustrating interference ofsurface hybridization of ligated (circularized) MIPs by unligated MIPprobes, e.g., in an assay mixture.

FIG. 42 provides graphs comparing the effects of treating assay mixturesto remove unligated MIP probes prior to hybridizing ligated,circularized MIPs to a surface. These data show the spots counted on asurface after hybridization and rolling circle signal amplification asdescribed herein. These data illustrate that inhibition by excessunligated MIP proves is reduced by treatment of the mixture with acombination of Exo I, Exo VII, recJ_(f) prior to hybridizing the productto immobilized primers on a surface. Treatment with Exo I alone showedsubstantial inhibition of circularized MIP hybridization, resulting inlow spot counts. Treatment with Exo I in combination with Exo III alsoshowed substantial inhibition of circularized MIP hybridization (datanot shown).

FIG. 43 provides a graph comparing the results of pretreatment of thereaction mixture with an original EXO reaction to the results using animproved nuclease digestion treatment as described herein, as reflectedin spot counts indicative of hybridization of circularized MIP probes.

FIG. 44 provides a schematic diagram illustrating use of MIP probes forquantitation of methylation of C residues, e.g., in CpG dinucleotideloci of a hyper- or hypomethylated gene or gene control region. In theillustrated embodiments, unmethylated cytosines are converted todeoxyuracils by treatment with a bisulfite reagent (e.g., sodiumbisulfite), such that the methyl C bases can be distinguished fromuracils bases using MIP technology. In alternative embodiments,Tet-assisted pyridine borane system is used, in which methylated DNA istreated with ten-eleven translocation (Teti) enzyme, which oxidizes both5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to5-carboxylcytosine (5caC). Pyridine borane is then used to reduce 5caCto dihydrouracil, a uracil derivative. Like the uracil in bisulfiteconverted DNA, the uracil is replaced by a thymine when the DNA isreplicated, e.g., in a polymerase chain reaction. (See, e.g., Liu Y, etal., Nat Biotechnol. 2019 April; 37(4):424-429.

FIG. 45 illustrates primers immobilized via a 5′ terminal aminemodification. In the embodiment shown, the primer 3′ ends contain threedue bases and an AlexaFluor488 tag at the 3′ terminus. The immobilizedprimers are treated with a mixture of uracil DNA glycosylase and DNAglycosylase-lyase Endonuclease VIII (abbreviated USER) to converturacils to abasic sites and to cleave of the abasic sites. The resulting3′ end is polymerase extendible. The conversion of the immobilizedoligonucleotides into extendible primers can be monitored by monitoringfluorescence released from the oligonucleotides, allowing measurementoligonucleotide immobilization and final primer density.

FIG. 46 illustrates primers immobilized via a 5′ terminal aminemodification, hybridized to oligonucleotides labeled with a fluorophore(e.g., AlexaFluor488, as shown). After hybridization of the fluor-taggedoligonucleotides to the immobilized primers, excess taggedoligonucleotides are removed, and the plate is washed. The bound taggedoligonucleotides can be melted off, e.g., with NaOH, the solutionneutralized (e.g., with HCl) and the released fluorescence is measured(e.g., in a Spectramax plate reader). The embodiment illustrated allowsfor rapid characterization of hybridization conditions in a manner thatis independent of rolling circle amplification efficiency.

FIG. 47 provides graphs illustrating the correlation between the amountof oligonucleotide immobilized with the measured fluorescence using themethod described above for FIG. 46, showing excellent linearity, as wellas a limit of detection of less than 100 fmoles.

FIG. 48 provides a graph illustrating the effect of treating a surfacewith a solution comprising sodium dodecyl sulfate (SDS) after exposingthe bound RCA product to labeled probes.

FIG. 49A shows the effects of addition of a detergent (Saponin, Teepol,Ammonium Lauryl Sulfate or SDS addition on a 2-step protocol in which amolecular beacon probe and the detergent (without graphene oxide) areadded after the RCA reaction and incubated for 1 hour at 37° C., asdescribed below.

FIG. 49B shows that the effects of addition of a detergent (Saponin,Teepol, Ammonium Lauryl Sulfate or SDS) in a 2-step protocol.

FIGS. 50A and 50B compare the number of spots developed on a during RCAamplification from MIPs localized on a surface in either stillreactions, or when the surface is agitated to mix the reagents duringthe course of the amplification reaction.

FIG. 51 shows a TEM image of 10 nm iron oxide nanoparticles that aremodified with an amphiphilic polymer coating and to comprisingcarboxylic acid functional groups (Ocean NanoTech, San Diego, Calif.).The organic layers consist of a monolayer of oleic acid and a monolayerof amphiphilic polymer, and have an average thickness of 4 nm, such thatthe particles have an average diameter about 8 to 10 nm larger than theinorganic core particle size. FIG. 51 illustrates conjugation of aprimer oligonucleotide to an iron oxide nanoparticle.

FIG. 52 provides a schematic diagram illustrating the effect of usingsmall nanoparticles, e.g., nanoparticles of about 20 nm diameter,compared to using large nanoparticles, e.g., nanoparticles of greaterthan about 200 nm. As shown, large nanoparticles can interfere withimaging RCA products.

FIG. 53 provides a schematic diagram of an embodiment of the technologyin which primers attached to nanoparticles, e.g., small iron oxidenanoparticles, are used to prime RCA amplification to form RCA productattached to nanoparticles.

FIG. 54 provides a graph that illustrates that the presence of unlabelediron oxide nanoparticles (lacking primers) in an RCA reaction mixture donot inhibit RCA amplification (1), and primers attached to iron oxidenanoparticles can prime RCA from a circular template (e.g., a ligatedMIP) (2). The RCA reactions were visualized by hybridization ofmolecular beacon probes.

FIG. 55 shows microscope images of surfaces in which iron oxide beadsare localized to a surface and imaged with an IXM4 microscope. The tophalf shows assay wells containing RCA reactions with iron oxidenanoparticles in which the primers were not attached to thenanoparticles, and the bottom half of the figure shows assay wellscontaining RCA products synthesized using primers attached to iron oxidenanoparticles, in which the RCA products were magnetically localized tothe bottom of the well prior to imaging.

FIG. 56 provides a schematic diagram of an embodiment of the technologyin which primers comprise a capture sequence on the 5′ end and abiotinylated oligonucleotide block on the 3′ end. In the embodiment ofthe diagram, a primer oligonucleotide hybridizes to s circularized RCAtemplate in solution and the Phi29 polymerase removes the 3′ block andextends the primer to form an RCA product. Excess primers that are notextended retain the 3′ biotin and may be removed, e.g., withstreptavidin-coated beads. The RCA product is captured by hybridizationto an immobilized capture oligonucleotide e.g., on a plate or wellsurface for detection. The RCA product is hybridized with labeledprobes, e.g., molecular beacon probes, before or after capture on thesurface, the labeled, immobilized complex is washed and detected.

FIG. 57A shows primer oligonucleotides designed for the in-solution RCA,e.g., in the embodiment shown schematically in FIG. 56, and containingdifferent arrangements of spacers.

FIG. 57B shows primer oligonucleotides designed for use in on-supportRCA, e.g., in the embodiment shown schematically in FIG. 35, andcontaining different combinations of spacers and amine modifications.

FIG. 58 provides a graph comparing end-point signal measured forreactions performed in solution or on on-plate using the primers shownin FIGS. 57A and 57B.

FIG. 59 provides a graph comparing quantitative RCA (qRCA) curvesmeasured using the “in-sol” primers shown in FIG. 57A with qRCA usingthe control primer (AUP).

DETAILED DESCRIPTION OF THE INVENTION

A goal in molecular diagnostics has been to achieve accurate, sensitivedetection of analytes in as little time as possible with the leastamount of labor and steps as possible. One manner in which this isachieved is the multiplex detection of analytes in samples, allowingmultiple detection events in a single reaction vessel or solution.However, many of the existing diagnostic methods, including multiplexreaction, still require many steps, including sample preparation stepsthat add to the time, complexity, and cost of conducting reactions. Thepresent invention, in some embodiments, provides solutions to theseproblems by providing assay that can be conducted directly in unpurifiedor untreated biological samples (e.g., blood or plasma).

In some embodiments, the technologies provided herein provide economicalmethods for testing samples in a manner that counts the number of copiesof a specific nucleic acid or protein in a sample or portion of a samplein a digital manner, i.e., by detecting individual copies of themolecules, without use of a sequencing step (e.g., a digital or “nextgen” sequencing step). The technologies find use for measuring targetmolecules such as nucleic acid molecules in any kind of sample,including but not limited to, e.g., samples collected for from a subjectfor diagnostic screening. Embodiments of the technology provided hereinfind use in, for example, non-invasive prenatal testing (NIPT) and othergenetic analysis. Embodiments of the technology implement one or moresteps of nucleic acid extraction, MIP probe design, MIPamplification/replication, and/or methods for measuring signal fromcircularized MIPs. In preferred embodiments, the technology providesmethods for immobilizing MIPs on a surface and detecting immobilizedMIPs. In preferred embodiments, immobilized MIPs are detected usingrolling circle amplification.

In preferred embodiments, the methods of the technology comprise atarget-recognition event, typically comprising hybridization of a targetnucleic acid, e.g., a sample of patient DNA, to another nucleic acidmolecule, e.g., a synthetic probe. In preferred embodiments, the targetrecognition event creates conditions in which a representative productis produced (e.g., a probe oligonucleotide that has been extended,ligated, and/or cleaved), the product then being indicative that thetarget is present in the reaction and that the probe hybridized to it.

A number of different “front-end” methods for recognizing target nucleicacid and producing a new product are described herein. For example, asshown in the exemplary embodiments in the Figures, the technologyprovides a number of ways to produce circularized molecules for use in a“back end” detection/readout step (see, e.g., FIGS., 1-3, 13-18, 34, 35,and 38-40). The technology also provides methods to signal the presenceof a target nucleic acid using other probe types, such as a probe thatcan be cleaved by a flap endonuclease in the presence of the targetnucleic acid (see, e.g., FIGS. 17-19). Each of these front-endembodiments can be used to produce a distinctive molecule, e.g., acircular or cleaved oligonucleotide.

These distinctive molecules may be configured to have one or morefeatures useful for capture and/or identification in a downstreambackend detection step. Examples of molecules and features produced in afront-end reaction include circularized MIPs having joined sequences(e.g., a complete target-specific sequence formed by ligation of the 3′and 5′ ends of the probe), having added sequences (e.g., copied portionsof a target template) and/or tagged nucleotides (e.g., nucleotidesattached to biotin, dyes, quenchers, haptens, and/or other moieties), orproducts such as single-stranded arms released from a flap cleavagereaction (see, e.g., FIGS. 17-19). In some embodiments, the MIPscomprise a feature in a portion of the probe, e.g., in the backbone ofthe probe.

Examples of back-end analysis methods for amplifying and/or detectingthe representative products of the front-end are provided, e.g., inFIGS. 2-3, 6-7, 9-12, 15-16, 20-21, 34, 35, and 38-41, 44-46, and 52

Although the technology is discussed by reference to particularembodiments, such as combinations of certain front-end target-dependentreactions with particular back-end signal amplification methods anddetection platforms, e.g., biotin-incorporated MIP of FIGS. 13-16coupled with an enzyme-free hybridization chain reaction back-end;biotin-tagged cleaved flaps (as in FIG. 19) coupled with capture to asurface, followed by hybridization to an enzyme-linked probe thatproduces fluorescence signal catalytically (as shown in FIG. 20), theinvention is not limited to the particular combinations of front-end andback-end methods and configurations disclosed herein, or to anyparticular methods of detecting a signal from the assay products. Itwill be appreciated that the skilled person may readily adapt onefront-end to work with an alternative back-end. For example, thecircularized MIP of FIG. 14 may be captured and detected using theenzyme-linked probe of FIG. 20, or might alternatively be amplified in arolling circle amplification assay, exemplified in FIGS. 2-3, 8-7, 9-12,21, 34, 35, and 38-41, 44-46, and 52. Similarly, the cleaved flap asshown in FIG. 19 may be detected using a hybridization chain reaction,as depicted in FIGS. 19-20; and a circularized MIP or an RCA ampliconmay be detected using an invasive cleavage reaction as diagrammed inFIG. 17, and so forth.

Further, although the technology is discussed in reference to particulartarget nucleic acids, e.g., cell-free DNA in plasma, the invention isnot limited to any particular form of DNA, or to any particular type ofnucleic acid, or to any particular type of variation in a nucleic acid.It will be appreciated that the skilled person may readily configureembodiments of the technology for detecting and counting mutations,insertions, deletions, single nucleotide polymorphisms (SNPs), andepigenetic variations in methylation (e.g., variations in methylation ofparticular CpG dinucleotides by analysis of DNA treated with a reagentthat converts unmethylated cytosines to uracils, thereby creatingdetectable sequence variations that reflect cytosine methylationvariations in target DNAs).

In some embodiments, assays are performed in a multiplexed manner. Insome embodiments, multiplexed assays can be performed under conditionsthat allow different loci to reach more similar levels of amplification.

FIG. 1 provides a schematic diagram of a molecular inversion probe(MIP). The molecular inversion probe contains first and second targetingpolynucleotide arms that are complementary to adjacent or proximalregions on a target nucleic acid to be detected, with a polynucleotidelinker or “backbone” connecting the two arms (see FIG. 1).

In the presence of a complementary target nucleic acid, the MIP can becircularized to form a MIP replicon suitable for detection. In someembodiments, the MIP is simply ligated using a nick repair enzyme, e.g.,T4 DNA ligase, AMPLIGASE thermostable DNA ligase, etc., while in someembodiments closing of the probe to form a circle comprises additionalmodification of the probe to create a ligatable nick, e.g., cleavage ofan overlap between the termini, filling of a gap between the terminiusing a nucleic acid polymerase, etc.

A target site or sequence, as used herein, refers to a portion or regionof a nucleic acid sequence that is sought to be sorted out from othernucleic acids in the sample that have other sequences, which isinformative for determining the presence or absence of a geneticdisorder or condition (e.g., the presence or absence of mutations,polymorphisms, deletions, insertions, aneuploidy etc.). A control siteor sequence, as used herein, refers to a site that has known or normalcopy numbers of a particular control gene. In some embodiments, thetargeting MIPs comprise in sequence the following components: firsttargeting polynucleotide arm—first unique targeting moleculartag—polynucleotide linker—second unique targeting molecular tag—secondtargeting polynucleotide arm. In some embodiments, a target populationof the targeting MIPs are used in the methods of the disclosure. In thetarget population, the pairs of the first and second targetingpolynucleotide arms in each of the targeting MIPs are identical and aresubstantially complementary to first and second regions in the nucleicacid that, respectively, flank the target site. See, e.g., WO2017/020023 and WO 2017/020024, each of which is incorporated herein byreference in its entirety.

In some embodiments, the length of each of the targeting polynucleotidearms is between 18 and 35 base pairs. In some embodiments, the length ofeach of the targeting polynucleotide arms is 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs, or any sizerange between 18 and 35 base pairs. In some embodiments, the length ofeach of the control polynucleotide arms is between 18 and 35 base pairs.In some embodiments, the length of each of the control polynucleotidearms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, or 35 base pairs, or any size ranges between 18 and 35 base pairs.In some embodiments, each of the targeting polynucleotide arms has amelting temperature between 55° C. and 70° C. In some embodiments, eachof the targeting polynucleotide arms has a melting temperature at 56°C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65°C., 66° C., 67° C., 68° C., 69° C., 70° C., or any temperature between55° C. and 70° C. In some embodiments, each of the controlpolynucleotide arms has a melting temperature between 55° C. and 70° C.In some embodiments, each of the control polynucleotide arms has amelting temperature at 56° C., 57° C., 58° C., 59° C., 60° C., 61° C.,62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C.,or any temperature between 55° C. and 70° C.

In some embodiments, each of the targeting polynucleotide arms has a GCcontent between 20% and 80%. In some embodiments, each of the targetingpolynucleotide arms has a GC content of 20-30%, 30-40%, or 30-50%, or30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or50-80%, or any range of GC content between 20% and 80%, or any specificpercentage between 20% and 80%. In some embodiments, each of the controlpolynucleotide arms has a GC content between 20% and 80%. In someembodiments, each of the control polynucleotide arms has a GC content of20-30%, 30-40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%,or 50-60%, or 50-70%, or 50-80%, or any range of GC content between 20%and 80%, or any specific percentage between 20% and 80%.

In some embodiments, the polynucleotide linker is not substantiallycomplementary to any genomic region of the sample or the subject. Insome embodiments, the polynucleotide linker has a length of 30 to 40base pairs. In some embodiments, the polynucleotide linker has a lengthof 31, 32, 33, 34, 35, 36, 37, 38, or 39 base pairs, or any intervalbetween 30 and 40 base pairs, and including 30 or 40 base pairs. In someembodiments, the polynucleotide linker has a melting temperature ofbetween 60° C. and 80° C. In some embodiments, the polynucleotide linkerhas a melting temperature of 60° C., 65° C., 70° C., 75° C., or 80° C.,or any interval between 60° C. and 80° C., or any specific temperaturebetween 60° C. and 80° C. In some embodiments, the polynucleotide linkerhas a GC content between 40% and 60%. In some embodiments, thepolynucleotide linker has a GC content of 40%, 45%, 50%, 55%, or 60%, orany interval between 40% and 60%, or any specific percentage between 40%and 60%.

In some embodiments, targeting MIPs replicons are produced by: i) thefirst and second targeting polynucleotide arms, respectively,hybridizing to the first and second regions in the nucleic acid that,together, form a continuous target site; and ii) after thehybridization, using a ligation reaction mixture to ligate the nickregion between the two targeting polynucleotide arms to formsingle-stranded circular nucleic acid molecules. In other embodiments,targeting MIPs replicons are produced by: i) the first and secondtargeting polynucleotide arms, respectively, hybridizing to the firstand second regions in the nucleic acid that, respectively, flank thetarget site; and ii) after the hybridization, using a ligation/extensionmixture to extend and ligate the gap region between the two targetingpolynucleotide arms to form single-stranded circular nucleic acidmolecules.

In certain embodiments, the methods described herein are used to detectexonic deletions or insertions or duplication. In some embodiments, thetarget site (or sequence) is a deletion or insertion or duplication in agene of interest or a genomic region of interest. In some embodiments,the target site is a deletion or insertion or duplication in one or moreexons of a gene of interest. In some embodiments, the target multipleexons are consecutive. In some embodiments, the target multiple exonsare non-consecutive. In some embodiments, the first and second targetingpolynucleotide arms of MIPs are designed to hybridize upstream anddownstream of the deletion (or insertion, or duplication) or deleted (orinserted, or duplicated) genomic region (e.g., one or more exons) in agene or a genomic region of interest. In some embodiments, the first orsecond targeting polynucleotide arm of MIPs comprises a sequence that issubstantially complementary to the genomic region of a gene of interestthat encompasses the target deletion or duplication site (e.g., exons orpartial exons).

Circular DNA molecules such as ligated MIPs are suitable substrates foramplification using rolling circle amplification (RCA). In certainembodiments of RCA, a rolling circle replication primer hybridizes to acircular nucleic acid molecule, e.g., a ligated MIP, or circularizedcfDNA. Extension of the primer using a strand-displacing DNA polymerase(e.g., φ29 (Phi29), Bst Large Fragment, and Klenow fragment of E. coliPol I DNA polymerases) results in long single-stranded DNA moleculescontaining repeats of a nucleic acid sequence complementary to the MIPcircular molecule.

In some embodiments, ligation-mediated rolling circle amplification(LM-RCA), which involves a ligation operation prior to replication, isutilized. In the ligation operation, a probe hybridizes to itscomplementary target nucleic acid sequence, if present, and the ends ofthe hybridized probe are joined by ligation to form a covalently closed,single-stranded nucleic acid. After ligation, a rolling circlereplication primer hybridizes to probe molecules to initiate rollingcircle replication, as described above. Generally, LM-RCA comprisesmixing an open circle probe with a target sample, resulting in anprobe-target sample mixture, and incubating the probe-target samplemixture under conditions promoting hybridization between the open circleprobe and a target sequence, mixing ligase with the probe-target samplemixture, resulting in a ligation mixture, and incubating the ligationmixture under conditions promoting ligation of the open circle probe toform an amplification target circle (ATC, which is also referred to anRCA replicon). A rolling circle replication primer (RCRP) is mixed withthe ligation mixture, resulting in a primer-ATC mixture, which isincubated under conditions that promote hybridization between theamplification target circle and the rolling circle replication primer.DNA polymerase is mixed with the primer-ATC mixture, resulting in apolymerase-ATC mixture, which is incubated under conditions promotingreplication of the amplification target circle, where replication of theamplification target circle results in formation of tandem sequence DNA(TS-DNA), i.e., a long strand of single-stranded DNA that contains aconcatemer of the sequence complementary to the amplification targetcircle.

In the embodiment illustrated in FIG. 2, circularized molecules A, B, C,and D consist of MIPs that are specific to chromosome 13, 18, 21, X,and/or Y, or to a reference chromosome such as Chr. 1. The sequence ofthe MIP surrounding the gap complements region of the targetedchromosome, and the backbone of the MIP contains a specific sequencethat is used to hybridize a probe that will contain a specificfluorescent dye (FITC, ALEXA, Dylight, Cyan, Rhodamine dyes, quantumdots, etc.). Step 1 comprises hybridizing the MIPs to cfDNA, a singlebase pair extension (or longer extension), and ligation to circularizethe extended MIP. Step 2 comprises rolling circle amplification of thecircularized MIP so that the sequence required to hybridize to thefluorescently labeled oligonucleotide is amplified. A*, B*, C*, D* arethe complement of the MIP sequence. Step 3 comprises hybridizing thefluorescently labeled probe to the rolling circle product. In theembodiment illustrated in FIG. 3, detection of the RCA product isfacilitated by molecular probes instead of fluorescent dye labeledoligonucleotides.

There are multiple ways to immobilize the MIP to a surface (e.g., a beador glass surface) For example, this may be accomplished by priming therolling circle amplification with a modified oligonucleotide comprisinga bindable moiety. Groups useful for modification of the primingoligonucleotide include but are not limited to thiol, amino, azide,alkyne, and biotin, such that the modified oligonucleotides can beimmobilized using appropriate reactions, e.g., as outlined in Meyer et.al., “Advances in DNA-mediated immobilization” Current Opinions inChemical Biology, 18:8: 8-15 (2014), which is incorporated herein byreference in its entirety, for all purposes.

Imaging of the fluorescent dye incorporated MIPs can be accomplished byusing methods comprising immobilization of MIPs to a surface (e.g.,glass slide or bead), e.g., using modifications of the MIP backbone tocontain modified bases that can be immobilized using appropriatereactions as outlined above and in Meyer et. al., supra. and detectedusing an antibody. Once immobilized to a surface, an antibody directedto an incorporated tag can be used to form antibody-MIP complexes thatcan be imaged with microscopy. In some embodiments, the antibody may beconjugated to enhance or amplify detectable signal from the complexes.For example, conjugation of β-galactosidase to the antibody allowsdetection in a single molecule array (“SIMOA”), using the processdescribed by Quanterix, wherein each complex is immobilized on a beadsuch that any bead has no more than one labeled immunocomplex, and thebeads are distributed to an array of femtoliter-sized wells, such thateach well contains, at most, one bead. With addition ofresorufin-β-galactopyranoside, the β-galactosidase on the immobilizedimmunocomplexes catalyzes the production of resorufin, which fluoresces.Upon visualization, the fluorescence emitted in wells having animmobilized individual immunocomplexes can be detected and counted. See,e.g., Quanterix Whitepaper 1.0, Scientific Principle of Simoa (SingleMolecule Array) Technology, 1-2 (2013); and Quanterix Whitepaper 6.0,Practical Application of Simoa™ HD-1 Analyzer for UltrasensitiveMultiplex Immunodetection of Protein Biomarkers, 1-3 (2015), each ofwhich is incorporated herein by reference for all purposes In someembodiments, the antibody-MIP complex may be directly detected, e.g.,using a solid state nanopore with an antibody labeled with poly(ethyleneglycol) at various of molecular weights, as described in Morin et. al.,“Nanopore-Based Target Sequence Detection” PLOS One,DOI:10.1371/journal.pone.0154426 (2016), incorporated herein byreference.

FIG. 4 provides a schematic diagram of an embodiment of the technologycomprising circularizing circulating cfDNA (ccfDNA) using asingle-strand ligase (e.g., CircLigase™ thermostable RNA ligase) to make“native circles” for detection. Once created, the circular ccfDNA may bedetected using a number of different methods, including a number of RCAmethods. For example, as diagrammed in FIG. 5, one embodiment of thetechnology comprising circularizing cfDNA and using “Golden GateAssembly” to add segments for detection (see, e.g., Engler, C., Kandzia,R., and Marillonnet, S. (2008) PLoS ONE 3, e3647.)

FIG. 6 illustrates an additional method of detecting ccfDNA. In thisembodiment, plasma samples are processed to purify ccfDNA, as previouslydescribed (see, e.g., M. Fleischhacker, et al., Methods for isolation ofcell-free plasma DNA strongly affect DNA yield, Clin Chim Acta. 2011Nov. 20; 412(23-24):2085-8). In Step 1, ccfDNA is heat denatured andtreated with T4 polynucleotide kinase to create 5′ phosphorylated and 3′hydroxyl end DNA fragments. Additional DNA repair, such as with T4 DNApolymerase, may be used to repair DNA before heat denaturation and T4polynucleotide kinase treatment. A complementation oligonucleotide witha 3′ protected end (so that it will not be extended by a polymerase) ishybridized to the ccfDNA. This complementary oligonucleotide consists ofchromosome specific regions, A and C, and a universal sequence, B.ccfDNA is extended and ligated to complete the circular DNA molecule.Circularized ccfDNA is purified from the oligonucleotide and RCA is usedby annealing an oligonucleotide to the universal sequence, B. After RCA,fluorescently labeled probes are hybridized to the rolling circleproduct.

FIG. 7 illustrates another method of detecting ccfDNA. Plasma samplesare processed to purify ccfDNA as previously described. Step 1, ccfDNAis heat denatured. A complementation oligonucleotide with aphosphorylated 5 prime protected end is hybridized to the ccfDNA. Thiscomplementary oligonucleotide consists of chromosome specific regions, Aand C, and a universal sequence, B. Both the ccfDNA and complimentaryoligonucleotide is extended. However, only the complimentaryoligonucleotide has a 5′ phosphate to allow completion of a circular DNAmolecule. Circularized complimentary oligonucleotide is amplified byrolling circle amplification using a primer complementary to theuniversal sequence, B. After rolling circle amplification, fluorescentlylabeled probes are hybridized to the rolling circle product.

FIG. 8 shows a schematic diagram of a synthetic circular DNA useful as atemplate for rolling circle amplification, and comprising bindingrolling circle primer-binding site and two probe binding sites, and anoptional binding moiety (e.g., biotin).

FIG. 9 provides a schematic diagram of an embodiment of the technologycomprising use of pairs of probes configured for collisional quenchingwhen hybridized to a strand of DNA, for use in detection of product fromRCA, e.g., of a circular DNA like the one shown in FIG. 8. In thisembodiment, the dye-labeled probe in solution is not quenched, andproduces signal. Probes hybridizing to the target near quencher-taggedprobes would be quenched, thereby reducing the fluorescence signal. Asthe amount of RCA product increases, the fluorescence decreases.

FIG. 10 provides a schematic diagram of an embodiment of the technologycomprising use of pairs of probes configured for fluorescence resonanceenergy transfer (FRET), as described above, when hybridized to a strandof DNA, for use in detection of product from RCA.

FIG. 11 provides a schematic diagram of an embodiment of the technologycomprising use of probes comprising a dye and a quencher, configured tobe cleaved, e.g., using a duplex-specific nuclease, such as arestriction enzyme, when hybridized to a strand of DNA, for use indetection of product from RCA.

As diagrammed in FIG. 12, one embodiment of the technology comprisinguse of RCA of chromosome-specific identifier sequences (CIDs), followedby CID-specific digestion of non-targeted chromosomes, and CID-specificlabeling directed to targeted CIDs. CIDs are amplified by RCA butmaintain their individual single molecule identities. CID amplificationincreases the fluorescence signal from individual target molecules.Sequences from chromosomes that are not being analyzed aredually-repressed by enzymatic digestion and the use of labels specificonly for the chromosomes being analyzed.

In some embodiments, a MIP may be detected using non-enzymatic method ofsignal amplification. For example, in some embodiments, a MIP isimmobilized on a surface, and is detected using a method such as atriggered “hybridization chain reaction” (HCR), e.g., as described by RM Dirks, et al., Proc. Natl. Acad. Sci. USA 101(43):15275-15278 (2004),and U.S. Pat. No. 8,105,778, each of which are incorporated herein byreference. FIGS. 13-16 illustrate an exemplary configuration using HCRfor signal amplification.

FIG. 13 illustrates an embodiment in which MIPs hybridize to targetnucleic acid, e.g., cfDNA, leaving a single nucleotide gap. The gap isfilled by extension to incorporate a biotinylated nucleotide and closedby ligation. The circularized MIPs may then be bound to astreptavidin-coated surface, as illustrated in FIG. 14, and, afterwashing away any unbound MIPs, the backbone of the bound MIP ishybridized to an initiator oligonucleotide. In preferred embodiments, aspacer, e.g., an 18-atom hexa-ethyleneglycol spacer, is included betweenthe initiator sequence and backbone-binding sequence. Preferably, thefootprint of the MIP binding region is selected to have a high T_(m)(e.g., approx. 79° C.), for stable binding. As discussed above, bindingtags are than biotin, such as an amine group, a thiol group, an azide,or a hapten, may be used to tag and immobilize the MIP to anappropriately reactive surface.

FIG. 15 shows examples of hairpin oligonucleotides used in the HCR toform a self-assembling scaffold. One or both oligonucleotides comprisesat least one label, e.g., a fluorophore. In preferred embodiments, thedyes are positioned to provide a sufficiently large spacing in theassembled scaffold to prevent quenching effects. For example, in someembodiments, the dyes are positioned on opposite ends of the hairpins,as shown in FIG. 14. As shown in FIG. 16, once the reaction is initiatedby hybridization to the initiator oligonucleotide bound to the MIPbackbone, the HCR hairpins unfold and hybridize in in long strands,creating a scaffold comprising a large number of labels.

A flap endonuclease reaction (e.g., Invader assay) may be used forspecific, quantitative detection of chromosomes. An exemplary embodimentis illustrated in FIGS. 17-20. FIG. 17 shows an Invader oligonucleotideand a probe oligonucleotide hybridized to a target region of achromosome. The 3′ end of the invasive oligonucleotide overlaps with the5′ end of the region of the probe oligonucleotide that is complementaryto the target region. In this embodiment, the probe oligonucleotidecomprises a 5′ flap comprising a biotin moiety, and a 3′ tail comprisinga label, e.g., a fluorophore. A flap endonuclease, e.g., a FEN-1nuclease, recognizes the overlapping invasive cleavage structure andcleaved the probe in a highly specific, structure-dependent manner,releasing the 5′ flap. In preferred embodiments, the reaction is runisothermally and produces linear signal amplification, providing 10³ to10⁴ cleaved probes per target in one to three hours, as shownschematically in FIG. 19.

In preferred embodiments, the probe oligonucleotide used comprises ahairpin structure in which the 5′ flap and the 3′ tail of the probehybridize to each other, as illustrated in FIG. 18. The fluorophore oranother moiety, e.g., 2,4 dinitrophenyl, may be used as haptens, suchthat uncleaved probes and/or the 3′ portions of the cleaved probes maybe removed from the reaction using an antibody to the hapten forcapture.

Cleaved flaps from the flap endonuclease reaction may be detected in anumber of ways. In a preferred embodiment, the cleaved flap is capturedusing an immobilized complementary probe, and the biotin is reacted withstreptavidin linked to a detectable moiety, as illustrated in FIG. 20.In the embodiment shown, the streptavidin is coupled to β-galactosidase,and a fluorescence signal is generated by providing non-fluorescentresorufin-β-galactopyranoside, which is catalyzed by the β-galactosidaseto produce the D-galactose and the fluorescent dye resorufin. Usingfemtoliter arrays and Poisson statistics to produce a digital readoutforma, single hybridization events can be detected using such enzymaticsignal amplification. See, e.g., D M Rissin and D R Walt, DigitalConcentration Readout of Single Enzyme Molecules Using Femtoliter Arraysand Poisson Statistics. Nano Letters 6(3):520-523 (2006); QuanterixWhitepaper 1.0, Scientific Principle of Simoa (Single Molecule Array)Technology, 1-2 (2013); and Quanterix Whitepaper 6.0, PracticalApplication of Simoa™ HD-1 Analyzer for Ultrasensitive MultiplexImmunodetection of Protein Biomarkers, 1-3 (2015), each of which isincorporated herein by reference for all purposes. In certain preferredembodiments a kinetic readout, i.e., collecting signal from the array attwo time points, is used.

In the embodiment illustrated in FIGS. 21, A, B, C, and D consist ofMIPs that are specific to chromosome 13, 18, 21, X, Y, or a referencechromosome such as 1. The sequence of the MIP surrounding the gapcomplements region of the targeted chromosome and is designed to containa single nucleotide gap. Step 1: This gap is filled in with a dNTPconjugated to a hapten such as a fluorescent dye, biotin, etc. Fillingthe gap introduces a different hapten into MIPs targeted to each of thedifferent specific chromosomes. For example, addition of an A completesonly MIPs targeted to chromosome 21, T completes MIPs targeted tochromosome 18, G completes MIPs targeted to chromosome 13, and Ccompletes MIPs targeted to a reference chromosome such as chromosome 1.This approach labels these four different MIPs with a four uniquehaptens. To increase the number of chromosomes investigated, samples canbe split into two or more samples. These split samples can be reactedwith different MIP pools to incorporate 8 or more different haptensusing the same concept explained above in two or more separatereactions. Pools of MIPs targeted to each chromosome requiring aspecific dNTP to complete the single extension and ligation are used toincrease the number of capture events. Step 2 comprises incubating thehapten-containing MIPs with labeled antibodies specific to each hapten.The labels may comprise, e.g., a fluorescent dye, quantum dot, or otherfluorescent particles. Step 3 comprises an optional step of exposing theimmunocomplexes comprising the hapten-targeted primary antibodies to alabeled secondary antibody directed against the primary antibody,thereby amplifying the fluorescent signal.

As illustrated in FIG. 21, in this embodiment of the technology, MIPsdesigned to target different chromosomes each require a differentnucleotide to extend and ligate, and wherein the MIPs are extended andligated in a chromosome-specific manner using nucleotides which carrydifferent dyes for each different dNTP. For example, in a preferredembodiment, CY2, CY3, CYS, and CY7 are used. The dye-tagged MIPs may bedetected using antibodies specific for each different dye (and, byextension, for each different chromosome to be detected). Signal can beamplified by the use of secondary antibodies. For example, CY2 primaryrabbit antibody is bound to the target MIP, and secondary goatanti-rabbit antibody is bound to primary antibody to amplify signal,etc.)

As discussed above, many different fluorescence labeling systems findapplication in the embodiments of the technology. In some embodiments,fluorescent dyes (e.g., fluorescein, Texas Red, TAMRA, Cy3, Cy5, may beused, e.g. attached to nucleotide analogs incorporated intooligonucleotides or extension products. In some embodiments, fluorescentparticles, e.g., nanoparticles, nanocrystals, quantum dots, silica(e.g., mesoporous silica nanoparticles) polymer beads (e.g., latex), maybe used.

Many options exist for detection and quantitation of fluorescence signalfrom the embodiments of the technology described hereinabove. Detectioncan be based on measuring, for example physicochemical, electromagnetic,electrical, optoelectronic or electrochemical properties, orcharacteristics of the immobilized molecule and/or target molecule. Twofactors that are pertinent to single molecule detection of molecules ona surface are achieving sufficient spatial resolution to resolveindividual molecules, and distinguishing the desired single moleculesfrom background signals, e.g., from probes bound non-specifically to asurface. Exemplary methods for detecting single molecule-associatedsignals are found, e.g., in WO 2016/134191, which is incorporated byreference herein in its entirety for all purposes. In some embodiments,assays are configured for standard SBS micro plate detection, e.g., in aSpectraMax microplate reader or other plate reader. While this methodtypically requires low-variance fluorescence (multiple wells, multiplemeasurements), this format can be multiplexed and read on multipledifferent fluorescence channels. Additionally, the format is very highthroughput.

Embodiments can also be configured for detection on a surface, e.g., aglass, gold, or carbon (e.g., diamond) surface. In some embodiments,signal detection is done by any method for detecting electromagneticradiation (e.g., light) such as a method selected from far-field opticalmethods, near-field optical methods, epi-fluorescence spectroscopy,confocal microscopy, two-photon microscopy, optical microscopy, andtotal internal reflection microscopy, where the target molecule islabelled with an electromagnetic radiation emitter. Other methods ofmicroscopy, such as atomic force microscopy (AFM) or other scanningprobe microscopies (SPM) are also appropriate. In some embodiments, itmay not be necessary to label the target. Alternatively, labels that canbe detected by SPM can be used. In some embodiments, signal detectionand/or measurement comprises surface reading by counting fluorescentclusters using an imaging system such as an ImageXpress imaging system(Molecular Devices, San Jose, Calif.), and similar systems.

Embodiments of the technology may be configured for detection using manyother systems and instrument platforms, e.g., bead assays (e.g.,Luminex), array hybridization, NanoString nCounter single moleculecounting device. See, e.g., G K Geiss, et al., Direct multiplexedmeasurement of gene expression with color-coded probe pairs; NatureBiotechnology 26(3):317-25 (2008), U.S. Patent Publication 2018/0066309A1 published Mar. 8, 2018, (P N Hengen, et. Al., Invent., NanostringTechnogies, Inc.), etc.

In the Luminex bead assay, color-coded beads, pre-coated withanalyte-specific capture antibody for the molecule of interest, areadded to the sample. Multiple analytes can be simultaneously detected inthe same sample. The analyte-specific antibodies capture the analyte ofinterest. Biotinylated detection antibodies that are also specific tothe analyte of interest are added, such that an antibody-antigensandwich is formed. Phycoerythrin (PE)-conjugated streptavidin is added,and the beads are read on a dual-laser flow-based detection instrument.The beads are read on a dual-laser flow-based detection instrument, suchas the Luminex 200™ or Bio-Rad® Bio-Plex® analyzer. One laser classifiesthe bead and determines the analyte that is being detected. The secondlaser determines the magnitude of the PE-derived signal, which is indirect proportion to the amount of bound analyte.

The NanoString nCounter is a single-molecule counting device for thedigital quantification of hundreds of different genes in a singlemultiplexed reaction. The technology uses molecular “barcodes”, each ofwhich is color-coded and attached to a single probe corresponding to agene (or other nucleic acid) of interest, in combination withsolid-phase hybridization and automated imaging and detection. See, e.g.Geiss, et al., supra, which describes use of unique pairs of capture andreporter probes constructed to detect each nucleic acid of interest. Inthe embodiment described, probes are mixed together with the nucleicacid, e.g., unpartitioned cfDNA, or total RNA from a sample, in a singlesolution-phase hybridization reaction. Hybridization results in theformation of tripartite structures composed of a target nucleic acidbound to its specific reporter and capture probes, and unhybridizedreporter and capture probes are removed e.g., by affinity purification.The hybridization complexes are exposed to an appropriate capturesurface, e.g., a streptavidin-coated surface when biotin immobilizationtags are used. After capture on the surface, an applied electric fieldextends and orients each complex in the solution in the same direction.The complexes are then immobilized in the elongated state and areimaged. Each target molecule of interest can thus be identified by thecolor code generated by the ordered fluorescent segments present on thereporter probe and tallied to count the target molecules.

FIG. 22, panels A, B, and C, illustrate embodiments of the technology inwhich MIPs comprising or attached to an immobilization moiety areimmobilized on a surface. While not limited to any particular embodimentfor incorporating a representaati feature indicative of targetrecognition into a circularized MIP molecule, the embodiments of FIG. 22are illustrated using an embodiment comprising extension of the linearMIP using a polymerase to copy one or more nucleotides of a targetnucleic acid, followed by ligation to circularize the extended probe.

In the embodiment illustrated in panel A of FIG. 22, in step 1 MIPs arehybridized with the target DNA and are then extended by a DNA polymerasein the presence of modified dNTPs so that an immobilization moiety isincorporated into each MIP during extension. The MIP is then ligated toitself to complete the circularized probe. The modified dNTPs maycomprise, but are not limited to, dNTPs comprising reactive chemistryspecies such as amine groups or thiol groups, or other bindablefeatures, such as biotin or an antibody hapten. In step 2, circularizedMIPs are exposed to a surface under conditions in which the surfaceinteracts with the immobilization feature of the MIP to bind the MIP.Such surfaces include but are not limited to derivatized orunderivatized glass, silica, diamond, gold, agarose, plastic,ferromagnetic material, alloys, etc., and may be in any form, e.g.,slide, sample well, channel, bead, particle and/or nanoparticles, any ofwhich may be porous or non-porous.

In the embodiment illustrated in panel B of FIG. 22, in step 1, MIPs arehybridized with target DNA and ligated to circularize. In the embodimentshown, the MIP is extended by a DNA polymerase to fill a sequence gapprior to ligation, while in other embodiments, the MIP may be designedto be simply hybridized to the target nucleic acid and ligated tocircularize without use of a polymerization step, in the manner, e.g.,of padlock probes See, e.g., M. Nilsson, et al. “Padlock probes:circularizing oligonucleotides for localized DNA detection”. Science.265 (5181): 2085-2088 (1994); J. Banér, et al., Nucleic Acids Res., 26(22):5073-5078 (1998). In step 2, the circular MIP is hybridized to acomplementary oligonucleotide that contains an immobilization moiety asdescribed above, e.g., a reactive amine, a reactive thiol group, biotin,a hapten, a capturable nucleic acid tag sequence, etc. In step 3, thehybrid MIP complex of the MIP and the oligonucleotide comprising theimmobilization moiety is exposed to a surface under conditions in whichthe surface interacts with the immobilization feature of the MIP complexto bind the MIP complex. As described above, surfaces include but arenot limited to derivatized or underivatized glass, silica, diamond,gold, agarose, plastic, ferromagnetic material, alloys, etc., and may bein any form, e.g., slide, sample well, channel, bead, particle and/ornanoparticles, any of which may be porous or non-porous.

In the embodiment illustrated in panel C of FIG. 22, in step 1, MIPsthat contain an immobilization moiety built into the backbone of theprobe are hybridized with DNA, extended by a DNA polymerase, and ligatedto circularize the probe. As with the embodiment of panel B describedabove, the MIPs may be designed to be simply hybridized to a targetnucleic acid and ligated to circularize without use of a polymerizationstep. In step 2, the circularized MIP containing the immobilizationmoiety is exposed to a surface under conditions in which the surfaceinteracts with the immobilization feature of the MIP to bind the MIP. Asdescribed above, surfaces include but are not limited to derivatized orunderivatized glass, silica, diamond, gold, agarose, plastic,ferromagnetic material, alloys, etc., and may be in any form, e.g.,slide, sample well, channel, bead, particle and/or nanoparticles, any ofwhich may be porous or non-porous.

In each of the embodiments illustrated in FIG. 22, once the MIPs havebeen immobilized to a surface, labeling and/or signal amplification(e.g., fluorescent labeling and/or amplification of fluorescent signal)and detection can be accomplished using any of the various back-endanalysis methods discussed herein. Suitable methods for amplifyingand/or detecting the unique immobilized MIP products include but are notlimited to the NanoString nCounter technology described above, and themethods illustrated in FIGS. 2-3, 6-7, 9-12, 15-16 and 20-21. In someembodiments, labeling and/or signal amplification (e.g., fluorescentlabeling and/or amplification of fluorescent signal) is done before theMIPs have been immobilized to a surface.

In preferred embodiments, a back-end process configured for singlemolecule visualization is used. For example, as described above, is theQuanterix platform uses an array of femtoliter-sized wells that capturebeads having no more than one tagged complex, with the signal from thecaptured complexes developed using aresorufin-β-galactopyranoside/β-galactosidase reaction to producefluorescent resorufin. Visualization of the array permits detection ofthe signal from each individual complex. In certain preferredembodiments, a solid state nanopore device, e.g., as described by Morin,et al., (see “Nanopore-Based Target Sequence Detection” PLoS ONE11(5):e0154426 (2016)), is used. A solid-state nanopore is a nano-scaleopening formed in a thin solid-state membrane that separates two aqueousvolumes. A voltage-clamp amplifier applies a voltage across the membranewhile measuring the ionic current through the open pore. When a singlecharged molecule such as a double-stranded DNA is captured and driventhrough the pore by electrophoresis, the measured current shifts, andthe shift depth (SI) and duration are used to characterize the event.(Morin, et al., supra). Although DNA alone is detectable using thissystem, distinctive tags (e.g., different sizes of polyethylene glycol(PEG)) may be attached to highly sequence-specific probes (e.g., peptidenucleic acid probes, PNAs) to give any particular DNA-PNA-PEG complex adistinctive signature that represents the target nucleic acid detectedin the front-end of the assay.

In the embodiment illustrated in FIG. 23, a complex is formed comprisingan oligonucleotide primer and a circular probe, such as a MIP or ligatedpadlock probe. Extension of the primer in a rolling circle amplificationreaction produces long strand of single-stranded DNA that contains aconcatemer of the sequence complementary to the circular probe. The RCAproduct binds to a plurality of molecular beacon probes having afluorophore and a quencher. Hybridization of the beacons separates thequencher from the fluorophore, allowing detection of fluorescence fromthe beacon. Accumulation of the RCA product may be monitored in realtime by measuring an increase in fluorescence intensity that isindicative of binding of the beacons to the increasing amount of productover the time course of the reaction.

Real-time quantitation of accumulating fluorescence in reactions wasused to examine the effects of attached biotin moieties on the MIP or onthe primer. FIGS. 24A-24D show results from examining the effect on RCAsignal of including biotin residues in the circularized MIP only (A), inthe RCA primer only (B), in both (C), and in neither (D). In thisexperiment, the MIP contained the sequence:

(circularized) (SEQ ID NO: 23) 5′-CCTCCCATCATATTAAAGGCCTCTATGTTAAG

GACCTACGACG ATGCTGCTGCTGTACTACGAGGCTAAGGCATTCTGCAAACAT-3′.

In the biotinylated MIP above, the boxed ‘T’ shows the site ofattachment of a biotin (Integrated DNA Technologies, “Internal BiotindT”) in the MIP containing a biotin. The biotinylated primer comprised abiotin attached at the terminal ‘5’ phosphate (Integrated DNATechnologies, “‘5’ Biotin-TEG”). The rolling circle reaction wasconducted according to the “standard rolling circle reaction” proceduredescribed below in Example 1, at 37° C. for one hour. These data showthat the presence of biotin in the circularized MIP inhibits RCA, whilethe presence of biotin on the primer does not inhibit the reaction.

FIGS. 25 A-C show the results of varying amounts of components instandard RCA reactions in solution. FIG. 25A compares use of 5 units and25 units of Phi29 polymerase in each reaction, and shows that the higherconcentration of polymerase yielded consistently higher signal under theconditions tested. FIG. 25B shows the effects of using differentconcentrations of molecular beacon probe (“Beacon”); FIG. 25C comparesthe effects of using the different concentrations of Phi29 polymeraseand molecular beacon probe to the effect on the standard reaction ofusing 200 μM or 800 μM total dNTPs. Based on these data, reactionsadjusted to comprise 1000 nM beacon, 800 μM dNTPs, and 2000 nM phi 29polymerase (80 units) were further tested.

The effects of adding different concentrations of PEG and of usingdifferent sizes of PEG to the enhanced RCA conditions (E-RCA, seeExample 1, below) were examined. FIG. 26 compares the effects of usingdifferent sizes of PEG (200 and 8000) at the percentages (w:v) shown, inthe E-RCA conditions. Under the conditions tested for this embodiment,PEG 200 provided superior results at all concentrations tested, with 20%PEG 200 providing the best results. In contrast, the PEG 8000significantly reduced the efficiency of the RCA. Based on these data,RCA reactions comprising at least 20% w:v of PEG 200 were furthertested.

As discussed above, single molecule detection on a surface, it ispreferable that the spot size of the signal from any individual boundmolecule be minimized, such that separation between spots is assuredsuch that individual spots can be resolved by a light microscope. Theeffects of using PEG 200 on the spot size and the number of spotsdetected was examined. The assays were conducted using the E-RCAconditions described below, with or without 20% w:v PEG 200, incubatedfor 140 min. The results are shown in FIGS. 27A-27B and 28A-28B. FIG.27A shows that the presence of PEG decreased the spot size, enhancingmeasurement of fluorescence signal from individual spots. FIG. 27B showsthe effects of PEG on the number and fluorescence intensity of the spotsshown in FIG. 27A, and shows that addition of PEG increased the numberof detectable spots while reducing the size of the spots detected.

The effects on spot count and spot size using different molecularweights of PEG in a 20% solution in reactions conducted onAPTES-silanized plates were examined. Reactions on the APTES-treatedsurface were conducted as described in the “One-Step Rolling CircleAmplification On a Surface” in Example 1, with the PEG componentmodified as indicated in FIG. 28. FIG. 28 shows that spot number ismaximized and spot size is minimized when the PEG used is smaller than1000, preferably between 200 and 800, more preferably 600 averagemolecular weight.

The length of hybridization time prior to initiating the RCA reactionwas examined. FIG. 29A shows microscope images of surfaces ofAPTES-silanized plates, as described in Example 1, and compares RCAsignal for reactions hybridized for 18 hours or 1 hour prior toinitiating the RCA reaction, in either TBS or RCA buffer The EnhancedRCA was performed as described above, with 20% PEG 600, for 140 minutes.FIG. 29B provides graphs comparing the effects of hybridization time andbuffer on the number and fluorescence intensity (area) of the spotsshown in FIG. 29A. These data show a substantial increase in the numberof spots when with longer hybridization time.

FIGS. 30, 31, and 32 provide graphs comparing the effects of PEG 200 onthe standard RCA reaction conditions, on the enhanced RCA (E-RCA)conditions, and on the E-RCA conditions with additional variations, withor without a 2 hour hybridization time, and with PEG 2000 in place ofPEG 200, with a 2 hour hybridization. The reactions in each figure wereall performed at the same temperature, with the reactions performed at30° C., 25° C., and 37° C. in FIGS. 30, 31, and 32, respectively.

The number and pixel size (area, in pixels) of the spots were assessedfor each condition. Using the IXM4 microscope, a single pixel width isapproximately 334 nm. These data show that in the presence of PEG 200,the 37° C. reaction temperature gave the best combination of high spotcount and small spot size. The effect of varying the concentrations ofbeacon probe using higher RCA reaction temperatures was also examined.Reactions containing 1000, 2000, 4000, or 8000 nM molecular beaconprobes were conducted at 37° C. or 45° C., and showed that at the highertemperature, the number of spots counted increased substantially (datanot shown). While not limiting the technology to any particularmechanism of action, these data suggest that conducting the reactions athigher temperature, e.g., 45° C. or above, results in more RCA productand more bound beacon probe.

The effect of increased temperature in the presence of varyingconcentrations of PEG 600 was further examined. FIG. 33 shows microscopeimages of surfaces of APTES-silanized plates, as described in Example 1,and compares RCA signal for reactions comprising PEG 600 at theindicated concentrations, performed at 37° C. or 45° C. These data showthat 45° C. reactions produced substantially higher spot counts, andthat 10 to 15% w:v PEG 600 at 45° C. produced the best combination ofspot count and spot size.

The effect of adding graphene oxide to the RCA surface-bound reactionswas examined. A two-step RCA procedure as described in Example 2 andshown schematically in FIG. 35, was developed. FIG. 36 shows microscopeimages of surfaces of APTES-silanized plates, as described in Example 1,and shows RCA signal for two-step reactions graphene oxide. The negativecontrol contained no input target and shows background from themolecular beacon probe. FIG. 37 provides a graph comparing spot countsfor RCA reactions done one step (no GO) or two steps (with or withoutGO), comparing reactions with 100 fmol of circularized MIP to reactionswith no circularized MIP. These data show that use of GO substantiallyreduces the number of background spots in the no-target controlreactions, improving the signal:background result in the assay.

The technology provided herein is not limited to counting of nucleicacid molecules, and may be applied, for example, to counting othermolecules, e.g., macromolecules such as proteins and carbohydrates.FIGS. 38-40 provide schematic diagrams of different capture complexesfor applications of embodiments of the technology to detection ofdifferent types of target molecules.

Generally, it is preferable to avoid loss of target material, e.g.,target cfDNA so as to maximize the sensitivity of assay reactions.Target molecules such as nucleic acid are often lost during purificationsteps. For example, when matrix binding is used for purification (e.g.,chaotrope-mediated binding to glass matrix prior to washing andelution), target material can be lost by incomplete capture from asample and/or incomplete release from the matrix during elution.Similarly, when precipitation is used, sample can be lost by incompleteprecipitation, or by incomplete dissolving after precipitation. Someembodiments of the present technology are configured to reduce oreliminate such purification steps between the steps of the assays. Inpreferred embodiments, the method is configured such that the fluidenvironment of each step is compatible with the fluid environment of thefollowing step. A fluid environment for a follow-on step may beconsidered compatible with a prior step if, for example, it uses thesame fluid environment (salts, buffer, detergents, reducing agents,e.g.,) or if the fluid environment of the prior step can be readilyadjusted to suit the follow-on step (e.g., by addition of a reagent,buffer, additive, etc., to modify the fluid environment, or by dilutionof all or part of the product of a prior step into a fluid environmentcompatible with the follow-on step). Accordingly, embodiments of thetechnology provided herein are preferably configured so that theproducts of each step flow to the next step without interveningpurification or isolation steps.

In some embodiments, unligated MIP probes used in a capture reaction caninhibit follow-on steps. FIG. 41, for example, illustrates the problemof unligated MIP probes interfering with hybridization of the circularligated MIPs to primers that are immobilized on a surface. Suchinhibition reduces the sensitivity of assays configured to detect andcount the ligated MIPs in an assay reaction. During development of thetechnology, it was determined that a combination of nuclease enzymescould be used to reduce or eliminate interference from the unligated MIPprobes. As shown in FIG. 42, the effects of treating the ligated MIPassay mixtures with a single exonuclease, Exo I, were compared to theeffects of treating the ligation mixture with a combination ofexonucleases prior to hybridizing the circularized MIPs to primersimmobilized on a surface. These data show the spots counted on a surfaceafter hybridization and rolling circle signal amplification as describedherein, and illustrate that inhibition of hybridization by excessunligated MIP is reduced by treatment of the mixture with thecombination of Exo I, Exo VII, and recJ_(f) prior to hybridizing theproduct to immobilized primers on a surface. Treatment with Exo I aloneshowed substantial inhibition of circularized MIP hybridization,resulting in low spot counts (granule counts). Based on these data, itwas determined that at least 1000-fold excess of MIP probes could beused, e.g., to drive efficient target binding and ligation, then theexcess could be removed by exonuclease treatment to remove inhibition ofprimer binding. After treatment with exonucleases, preferably with acocktail of exonucleases selected from, e.g., Exo I, Exo VII, recJ_(f),and/or Exo T, as described in Example 3, below, the nuclease enzymes areheat killed or otherwise disabled prior to exposing the nuclease-treatedMIP mixture to additional single-single stranded and linearoligonucleotides, such as primers or capture probes. In the experimentshown in FIG. 42, the enzymes were heat killed prior to binding of theligated MIPs to surface-immobilized primers. In other embodiments,nuclease may be selected such that 5′ conjugated primers (e.g.,conjugated to a nanoparticle or plate surface at the 5′ end) andcircular molecules such as MIPs are not substrates for the exonucleaseactivity. For example, these oligonucleotides are not substrate for flapnuclease or 5′ to 3′ exonucleases, which require a substrate to have afree 5′ end for digestion. In addition to the benefits described above,the ability to perform the ligation capture reactions in the presence ofa high concentration of MIP probe oligonucleotides also increases thecomplexity of multiplexing that can be performed using the technology.

FIG. 43 provides a graph comparing the results of pretreatment of thereaction mixture with an Exo I reaction to the results using a nucleasedigestion treatment comprising Exo I, Exo VII and recJ_(f), as describedherein, as reflected in spot counts indicative of hybridization ofcircularized MIP probes.

FIG. 44 provides a schematic diagram illustrating use of MIP probes forquantitation of methylation of C residues, e.g., in CpG dinucleotideloci of a hyper- or hypo-methylated gene or gene control region. In theillustrated embodiments, unmethylated cytosines are converted todeoxyuracils by treatment with a bisulfite reagent (e.g., sodiumbisulfite), such that the methyl C bases (C^(m)) can be distinguishedfrom uracil bases using MIP technology. As illustrated, MIP probes atmethylated C loci can be circularized by polymerase extension toincorporate a dG base, followed by ligation to close the resulting nickin the MIP strand.

In other embodiments, a Tet-assisted pyridine borane system is used, inwhich methylated DNA is treated with ten-eleven translocation (Teti)enzyme, which oxidizes both 5-methylcytosine (5mC) and5-hydroxymethylcytosine (5hmC) to 5-carboxylcytosine (5caC). Pyridineborane is then used to reduce 5caC to dihydrouracil, a uracilderivative. Like the uracil in bisulfite-converted DNA, dihydrouracil isrecognized as a T base during probe hybridization and is replaced by athymine when the modified DNA is replicated, e.g., in a polymerase chainreaction. (See, e.g., Liu Y, et al., Nat Biotechnol. 2019 April;37(4):424-429.) Application of the present technology to the counting ofmethylated and unmethylated loci in molecules may, for example, be usedto measure methylation levels without using sequencing methods, such asIllumina bisulfite sequencing. Target DNAs having CpG dinucleotides ofknown methylation levels (e.g., synthetic controls) can be used toestablish standard curves for determining methylation level as measuredby spot count. Measuring different degrees of methylation of DNAs findsapplication in characterizing a large number of disease states,including but not limited to cancers, imprinting disorders such asPrader-Willi disorder, and also in some embodiments, distinguishesbetween fetal and maternal DNAs.

Factors that influence immobilization and hybridization ofoligonucleotides (e.g., primers) include but are not limited to primerdensity (e.g., overly high density can lead to poor hybridization);electrostatic interference (e.g., repulsion between negative charges ofnucleic acid backbone, use of cations, e.g., sodium, potassium,magnesium, etc., to shield negative charges and reduce repulsion); anddistance of oligonucleotides from the surface (e.g., direct attachmentvs. use of spacer molecules to mimic solution kinetics). For example,higher concentrations of counterion (Na+) have been shown to yieldhigher densities of immobilized probes and faster immobilization, andheating of the probe film prior to hybridization has also been shown toincrease hybridization efficiency. See, e.g., Fuchs, J., et al.,Biophysical Journal Volume 99 September 2010 1886-1895; and Peterson, AW, et al, Nucleic Acids Res., 29(24): 5163-5168 (2001), each of which isincorporated herein by reference in its entirety, for all purposes.

During development of the technology, molecules were configured inseveral different ways in order to measure immobilization andhybridization on a surface. For example, as illustrated in FIG. 45, insome embodiments, primers are immobilized, e.g., via a 5′ terminal aminemodification (or other functional group for conjugation to a surface).In the embodiment shown, the primer 3′ ends contain three dU bases andan AlexaFluor488 tag at the 3′ terminus. The immobilized primers aretreated with a uracil-specific excision reagent, the USER enzymecocktail (New England Biolabs) to convert uracils to abasic sites and tocleave of the abasic sites. USER Enzyme is a mixture of Uracil DNAglycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDGcatalyzes the excision of a uracil base, forming an abasic(apyrimidinic) site while leaving the phosphodiester backbone intact.The lyase activity of Endonuclease VIII breaks the phosphodiesterbackbone at the 3′ and 5′ sides of the abasic site so that base-freedeoxyribose is released and a polymerase extendible 3′ OH is left. See,e.g., Lindhal, T., et al., (1977). J. Biol. Chem. 252, 3286-3294;Lindhal, T. (1982). Annu. Rev. Biochem. 51, 61-64; Melamede, R. J., etal., (1994). Biochemistry. 33, 1255-1264; and Jiang, D., et al., (1997).J. Biol. Chem. 272, 32230-32239. The conversion of the immobilizedoligonucleotides into extendible primers can be monitored by monitoringfluorescence released from the oligonucleotides, allowing measurementoligonucleotide immobilization and final primer density.

Hybridization to immobilized primers can be similarly tested byhybridization to fluorescently labeled test targets. As illustrated inFIG. 46, primers immobilized to a surface (e.g., via a 5′ terminal aminemodification) are hybridized to oligonucleotides labeled with afluorophore (e.g., AlexaFluor488, as shown). After hybridization of thefluor-tagged oligonucleotides, excess tagged oligonucleotides areremoved, and the plate is washed. The bound tagged oligonucleotides canbe melted off, e.g., with NaOH, the solution neutralized (e.g., withHCl) and the fluorescence released into the solution can be measured(e.g., in a Spectramax plate reader). The embodiment illustrated allowsfor rapid characterization of hybridization conditions in a manner thatis independent of rolling circle amplification efficiency, asillustrated in FIG. 47. The graphs in FIG. 47 show the correlationbetween the amount of fluorescently labeled oligonucleotide hybridizedto immobilized oligonucleotides using the method described above forFIG. 46, with the measured fluorescence showing excellent linearity, aswell as a limit of detection of less than 100 fmoles.

As discussed above, for single molecule detection on a surface, it ispreferable that the spot size of the signal (e.g., in pixel area ordiameter) from any individual bound molecule be minimized, such thatseparation between spots is assured and the spots can be resolved bymicroscopy, e.g., with a light microscope. FIG. 48 shows a comparison oftwo protocols. The rolling circle-graphene oxide protocol (rGO) usedgraphene oxide to quench the background false positive signal observedin the no-target (“no input”) control wells, but also decreased theoverall signal from all wells, including target-containing wells. Atwo-step protocol was used to examine the effect of treatment with SDSdetergent. In the first step, RCA was performed without fluorescentprobe. In the second step, the fluorescently labeled probeoligonucleotide was added in the presence of the detergent, in 1× Phi29polymerase buffer. These data show that the SDS detergent protocolreduced the no input false positive spots, but didn't decrease thesignal from other wells, and produced higher spot counts compared to thereactions without SDS. As shown in FIG. 48, the effects treating withSDS during the post-RCA staining step (with molecular beacon probesonly) were examined and compared to the graphene oxide protocol. Whilethe use of SDS had a similar effect on reducing the signal in thenegative control (“no input”) these data show that use of SDS producedhigher spot count compared to the use of RCA with molecular beacon stainand graphene oxide without a detergent step. We next tested multipleanionic detergents in this protocol. FIGS. 49A and 49B show that theeffects of using detergent in staining RCA products on a surface usingmolecular beacon probes in either a 2-step protocol (FIG. 49A) or a1-step protocol (FIG. 49B), as described hereinbelow. These data showthat inclusion of detergent reduced background in the no-target controlwhile not quenching fluorescence signal like the graphene oxide protocolon the samples having 1 or 10 fmol of input DNA (circular RCA template).

When the amplification is performed using a primer conjugated to a fixedsurface, e.g., a reaction well surface rather than a suspended beadsurface, the amplification reagents may be locally depleted during thereaction. Accordingly, embodiments of the technology, e.g., the 1-stepor 2-step reaction protocols described below, may include intermittentmixing steps (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, etc. minutes) to re-distribute reagents at time points throughoutthe reaction step incubations (e.g., during probe hybridization, rollingcircle amplification, fluorescent probe hybridization, etc.). Mixing isnot limited to any particular method and may comprising vortexing,bumping, rocking, tilting, or ultrasonic mixing. See, e.g., Ishigaki andSoto, Micromachines 2018, 9:272, which is incorporated herein byreference for all purposes. In FIG. 50A, the count of spots on a surfacegenerated with surface-immobilized primers for RCA, in reactionsperformed without shaking (“still”) or with intermittent shaking(“shaking”) are compared. These data show that intermittent mixingproduced a much larger number of detectable spots than the stillreactions produced.

During development of the technology, the effects of shaking ondifferent steps of the RCA assay were examined, as follows:

37° C. 60 min 25° C. 90 min 45° C. 90 min RCA + GO + 37° C. 60 min InputHyb RCA Beacon 5% Tween Exp 1 Shake Still Still Still Exp 2 Still ShakeStill Still Exp 3 Still Still Shake Still Ctrl 1 N/A Still N/A N/A Ctrl2 N/A Shake Still Still

Circular MIP oligonucleotides were hybridized to a surface-bound primerat 25° C. for 90 min. Rolling circle amplification was conducted at 45°C. for 90 min, and the RCA products were hybridized to fluorescentoligonucleotides in the presence of graphene oxide at 37° C. for 60 min.The products were washed with 5% Tween 20 at 37° C. for 60 min., thenmeasured for the number of countable spots at concentration. Control 1was a one step RCA protocol that was not shaken at any time and Control2 was a two step control RCA that was shaken during the RCA and heldstill during the fluorescent stain with the detergent. All conditionshad the same circularized MIPs as input. The shaking protocol incubatingthe RCA reactions at 45° C. for 90 minutes, with at-temperature mixingat 1500 rmp for the first 30 seconds of each 15 minute interval, asdescribed below in Example 5.

The results are shown in FIG. 50B. These data show that shaking the RCAstep provided the largest improvement in signal across different amountsof input MIPs.

The depletion effect may also be reduced or avoided when theamplification is performed in solution rather than on a fixed surface.Accordingly, in some embodiments, rolling circle amplification may beperformed in solution. In some embodiments the resulting product may belocalized to a support or surface after amplification, e.g., byhybridization to a probe that is bound to a surface, or by magnetic orcentrifugal manipulation of suspended to particles to bring them to asurface, such that detection is done with the products localized to asurface. FIG. 51 shows an example of iron oxide nanoparticles that arederivatized to provide carboxyl groups on the surface. As shownschematically, an oligonucleotide such as a primer may be linked to thenanoparticle via the reactive group.

Paramagnetic particles typically used for nucleic acid manipulations,e.g., for DNA purification or bead-emulsion PCR, are generally muchlarger than the 10 nm nanoparticles shown in FIG. 51. For example,DYNABEADS magnetic beads are >1 μm in diameter, and surface-activatedDYNABEADS comprising carboxyl or other reactive groups (ThermoFisherScientific, Inc.) come in 1 μm, 2.8 μm, and 4.5 μm sizes. However, asdiscussed above, products detected by microscopy, e.g., RCA productslabeled with fluorescent probes on the surface of a slide or well, arepreferably of a one or a few pixels in diameter, with each pixel beingapproximately 200 nm×200 nm for a light microscope, or 334 nm×334 nm forthe IMX4 microscope. Thus, as illustrated schematically in FIG. 52,beads having a diameter of more than about 200 nm can block fluorescencesignal from a product across one or more pixels, or can mask thepresence of RCA product entirely, making imaging difficult. Duringdevelopment of the technology it has been recognized that use of verysmall nanoparticles, e.g., paramagnetic particles that are less than apixel in diameter, preferably less than about 50 nm, more preferablyabout 20 nm in diameter, may be used in embodiments in which the RCAreaction (or another front-end reaction) may be performed on suspendednanoparticles, such that the reaction kinetics are more similar to areaction performed in solution, while the reaction product may bemanipulated magnetically or by, for example, centrifugation, to localizethe products at a surface, e.g., the bottom of a well, for back-enddetection, e.g., by microscopy. Use of the very small nanoparticles canreduce or eliminate masking of fluorescence signal by the particle. Inother embodiments, reaction products on particles may be measuredwithout such localization, using, e.g., flow cytometry.

In-Solution Amplification

In some embodiments, hybridization of ligated MIPs to immobilizedoligonucleotides (e.g., primers attached to a surface) may exhibitreduced efficiency, e.g., because of a lower effective concentration ofthe immobilized oligonucleotides. Additionally, the primer, MIP andenzyme and need to assemble at the surface, and surface charge and thedensity of the primers no the surface may also influence the reactionefficiency.

Accordingly, in some embodiments of the technology, oligonucleotides arehybridized to circularized MIPs in solution and are then localized orassociated with a surface, e.g., a surface of a well, either before orafter a further step, e.g., rolling circle amplification. In certainembodiments, surface-localized RCA products are detected by microscopy,as described hereinabove. Optionally, spacer molecules are included asmodifications to primers or to capture oligonucleotides complementary toprimers, such that immobilized primers or RCA products are further fromthe support surface, e.g., during primer extension and/or probehybridization.

FIG. 56 illustrates an embodiment of the technology detected byhybridization to an RCA primer in solution. In this embodiment, anin-solution primer (“in-sol” primer” is hybridized to a circularized MIPprobe (e.g., a padlock probe or other MIP probe that has beensuccessfully ligated after successful specific hybridization to a targetnucleic acid in a sample). In the embodiment shown, the primer comprisesan unextendible 3′ end, e.g., a nucleotide that is mismatched to the MIPtemplate, or that comprises a modification at the 3′ position of theribose. 3′ blocking nucleotides can be removed, e.g., by a 3′ to 5′exonuclease “proofreading” activity of a DNA polymerase such as Phi29polymerase, preferably in a template-dependent fashion, to render atemplate-bound primer extendible, while leaving unhybridized primeroligonucleotides blocked. In some embodiments, proofreading and primerextension are performed by a single enzyme, while in other embodiments,proofreading may be performed by a first enzyme, e.g., an autonomous3′-5′ exonuclease, and extension of the unblocked end may be performedby a second enzyme, a DNA polymerase. The technology is not limited toany particular form of 3′ block, and any form of nucleotide that lacks a3′ OH group required for extension by a DNA polymerase may be used.Examples of nucleotides lacking 3′ OH groups include dideoxy “chainterminator” nucleotides and nucleotides comprising a 3′ group such as a3′ phosphate, 3′ hexanediol (6-carbon chain), 3′ O-methyl, 3′ O-acyl, 3′O-allyl, 3′ O-ether, 3′ O-methoxymethyl, 3′ O-nitrobenzyl, 3′O-azidomethylene, 3′ C7-amine, or other substituent in place of the 3′OH group (see, e.g., D. Hutter, et al., Nucleosides Nucleotides NucleicAcids. 29(11):1-18 (2010), which is incorporated herein by reference inits entirety).

In the embodiment shown in FIG. 56, the 3′ end comprises a moiety forcapturing and removing excess primers. In some embodiments, the 3′ endof the primer comprises one or more biotins, (e.g., biotin dT, biotin ona triethyleneglycol (TEG) spacer, dual biotin), which can be used tocapture unextended primers, e.g., using streptavidin beads or the primermay comprise a hapten such as 2,4 dinitrophenol (2,4 DNP) ordigoxygenin, bindable by an antibody that recognizes the hapten.

EXPERIMENTAL EXAMPLES Example 1

This example provides examples of work-flows for analysis of DNA, e.g.,cfDNA, from a sample such as a blood sample.

Sample Collection

Blood is collected in a standard draw from patient. A 10 mL of bloodstored in a Streck blood collection tube or alternative EDTA-containingblood collection tube. The sample is transported into a lab at ambienttemperature and processed as follows:

-   -   Centrifuge blood at 2000×g for 20 minutes at room temperature to        obtain a plasma fraction from the blood.    -   Transfer plasma into a new, sterile, nuclease-free polypropylene        tube and centrifuge at 3220×g for 30 minutes.        Cell-Free DNA (cfDNA) Purification

Cell-free DNA is purified from plasma using standard methods, e.g.,using a MagMAX Cell-Free DNA isolation kit (Thermofisher Scientific,Cat. No. A29319).

Assay Plate Preparation

Glass bottom microtiter plates are treated to immobilize anoligonucleotide that primes the rolling circle amplification of acircularized MIPs. Several approaches can be used (see, e.g., E. J.Devor, et al., “Strategies for Attaching Oligonucleotides to SolidSupports,” Integrated DNA Technologies (2005), which is incorporatedherein by reference in its entirety, for all purposes.)

1) Acid Prewash

For each method, glass bottom plates are first acid washed as follows:

-   -   (a) Add 100 μL of 0.5 N sulfuric acid into each well.    -   (b) Add foil seal to plate.    -   (c) Incubate plate at 37° C. for 2 hours rotating at 300 RPM.    -   (d) Remove well contents.    -   (e) Wash wells twice with 100 μL molecular-grade water.    -   (f) Wash wells twice with 100 μL of 95% ethanol.

2) 3-Aminopropyltriethoxysilane (APTES) Silanization andStreptavidin-Biotin Primer Immobilization:

-   -   (a) Prepare 2% APTES by adding 200 μL 99% APTES (Sigma Aldrich,        Cat. No. 440140), 500 μL molecular-grade water, and 9.3 ml 95%        ethanol.    -   (b) Vortex solution and pipet 100 μL into each well.    -   (c) Incubate at room temperature for 15 minutes.    -   (d) Remove well contents.    -   (e) Wash wells twice with 100 μL of 95% ethanol.    -   (f) Remove last wash.    -   (g) Incubate plate at 37° C. for 24 hours.

Primer Immobilization

-   -   (h) To the amine-functionalized glass plates, add 1 nanogram of        streptavidin in 100 μL of Tris-buffered saline.    -   (i) Incubate at room temperature for 1 hour.    -   (j) Wash each well three times with 100 μL of TBS.    -   (k) Add 100 μL of a 1 μM solution of biotinylated        oligonucleotide.    -   (l) Incubate at room temperature for 1 hour.    -   (m) Wash each well three times with 100 μL of TBS.

3) Acrylic Silanization and Acrydite Primer Immobilization

-   -   (a) Prepare 4% acrylic silane by adding 400 μL 99% acrylic        silane (3-(Trimethoxysilyl)propyl methacrylate; Sigma Aldrich,        Cat. No. 440159), 1 mL molecular-grade water, and 18.6 mL 100%        ethanol.    -   (b) Add 100 μL of 4% acrylic silane solution to each well.    -   (c) Incubate at room temperature for 15 minutes.    -   (d) Remove 4% acrylic silane solution.    -   (e) Wash each well four times with 100 μL of 100% ethanol per        wash.    -   (f) Incubate plate at 37° C. for 24 hours.    -   (g) Prepare solution of acrydite-primer by adding        -   (i) 250 μL 5×TRIS Boron EDTA (TBE) buffer,        -   (ii) 500 μL 40% acrylamide,        -   (iii) 17.5 μL 10% ammonium persulfate,        -   (iv) 5 μL tetramethylethylenediamine (TEMED),        -   (v) 25 μL 100 μM oligonucleotide primer comprising a ‘5’            acrydite (or acrylic-phosphoramidite)        -   (vi) 1.7 mL of molecular-grade water.    -   (h) Add 25 μL of acrydite primer solution to each well and        gently agitate plate to cover well bottom.    -   (i) Incubate at room temperature for 30 min.    -   (j) Wash wells four times with 100 μL 0.5×TBE, discarding the        first three washes and leaving the last wash in well, before        continuing to RCA assay.

Primers may be immobilized by other methods, e.g., as described byDevor, et al., supra. In some embodiments, plates are treated with amonomer, e.g., dopamine or a derivative thereof, under conditionswherein a polymerized surface is formed and primers, e.g., primerscomprising a 5′ amine modification, are conjugated to the polymersurface.

In some embodiments, spacer of various lengths are used between thesurface and the oligonucleotide, to increase hybridization efficiency.In some embodiments, the hybridization efficiency is modified, e.g., bymodifying primer density on the surface, spacer length, salt,temperature hybridization, etc.

Molecular Inversion Probe Pool

A probe pool is used to capture specific loci in a DNA sample, e.g., acfDNA sample, and create circularized MIPs for rolling circleamplification. NIPT assays generally comprise a pool of molecularinversion probes. In preferred embodiments, a NIPT assay comprises about5,000-100,000 molecular inversion probes.

-   -   Targeted MIPs are created to target features to be investigated        by the assay (e.g., chromosomes 13, 18, 21, X, Y, and        CHR22q11.2).    -   Approximately 5,000 to 100,000 unique MIPs, preferably 5,000 to        50,000, preferably 10,000 to 40,000, preferably 20,000 to 30,000        unique MIPs are created for each feature.    -   MIPs are mixed together to create a probe pool with each probe        at a custom concentration.        MIP Capture of cfDNA and Ligation    -   MIP Pools are added to the purified cfDNA in the following        reaction.        -   2 μL of AMPligase Buffer (10×), 1 μL of a 10 μM MIP Probe            Pool, 16 μL of cfDNA prep, 1 μL of AMPligase (5 units), and            0.5 mM (final concentration) of NAD+ (β-Nicotinamide adenine            dinucleotide; New England Biolabs).        -   Reactions are incubated at 98° C. for 2 to 3 minutes and            cooled at 1 degree per minute until they reach 45° C., then            held for 2 hours at 45° C. In some embodiments, reactions            are cooled at 1 degree per minute to 56° C., held for 120            min., then reduced to 45° C. and held for an additional 120            min. In some embodiments, MIP oligonucleotides hybridized to            target regions in, e.g., cfDNA leave a gap in the MIP            strand. Thus, in some embodiments, MIP 3′ ends are extended,            e.g., with a polymerase, to fill the gap and produce a            ligatable nick.        -   In some embodiments, reactions are treated with an            exonuclease such as E. coli Exo I, to reduce the amount of            unligated MIP probe in the mixture. In preferred            embodiments, the technology provides treatment with a            nuclease or a cocktail of nucleases, e.g., one or more of            Exo I, Exo VII, Exo T, and recJ_(f) (New England Biolabs,            Inc., Ipswich, Mass.) prior to rolling circle amplification.            Exonuclease I (Exo I, E. coli) is a DNA-specific exonuclease            that catalyzes the removal of nucleotides from            single-stranded DNA in the 3′ to 5′ direction; Exonuclease            VII (Exo VII, E. coli) is a DNA-specific, single-strand            specific exonuclease that cleaves in both the 3′ to 5′ and            5′ to 3′ directions. Exonuclease T (or “RNase T”) catalyzes            removal of nucleotides from linear single-stranded DNA or            RNA in the 3′ to 5′ direction. RecJ_(f) is a single-stranded            DNA specific exonuclease that catalyzes the removal of            deoxynucleotide monophosphates from DNA in the 5′ to 3′            direction (Lovett, S. T., Kolodner, R. D. (1989). Proc.            Natl. Acad. Sci. USA. 86, 2627-2631.) RecJ_(f) is a            recombinant fusion protein of E. coli RecJ and maltose            binding protein (MBP). It has the same enzymatic properties            as wild-type RecJ, but fusion to MBP enhances RecJ_(f)            solubility.        -   In preferred embodiments the MIP ligation mixture is treated            with a cocktail of 2 or more of Exo I, Exo VII, Exo T,            and/or recJ_(f) nucleases prior to hybridization of the MIPs            with primers, e.g., surface immobilized primers. In            particularly preferred embodiments, the nuclease-treated MIP            ligation mixture is then applied to a surface without            intervening purification by, e.g., by spin column, matrix            binding, or precipitation. In particularly preferred            embodiments, the nucleases are neutralized, e.g., heat            killed prior to mixing the ligated, nuclease-treated MIP            mixture with additional nucleic acids, e.g., primers.            Molecular Beacon Probes

Examples of molecular beacon probes that find use in the technology areas follows:

(SEQ ID NO: 24) 5′Alexa 405-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-dabcyl 3′ (SEQ ID NO: 25) 5′Alexa 488-CCTCAATGCTGCTGCTGTACTACmGmAmGmG-dabcyl 3′(SEQ ID NO: 26) 5′Alexa 594-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ2 3′(SEQ ID NO: 27) 5′Alexa 647-CCTCAGCGCTGCCTATTCGAACTmGmAmGmG-BHQ2 3′ (SEQ ID NO: 28) 5′Alexa 750-CCTCAGGTGTGTAACTCGATCAGmGmAmGmG-BHQ3 3′These molecular beacon probes may be used individually or in multiplexcombinations.Fluorophore Labeled Probes

Examples of fluorescently labeled probes (without quencher moieties)that find use in the technology are as follows:

(SEQ ID NO: 29) 5′Alexa 488N/ATGCTGCTGCTGTACTAC 3′ (SEQ ID NO: 30)5′Alexa 546N/AGACAGCTAACTCAGACC 3′ (SEQ ID NO: 31)5′Alexa 594N/GGTGTGTAACTCGATCAG 3′ (SEQ ID NO: 32)5′Alexa 647N/CGCTGCCTATTCGAAC 3′ (SEQ ID NO: 33)5′Alexa 700N/CTGAAGTACCGCACGAAT 3′ (SEQ ID NO: 34)5′Alexa 750N/CATGGACGAGCTGTACAA 3′These fluorescently labeled probes may be used individually or inmultiplex combinations.Standard Rolling Circle Amplification Assay Conditions

-   -   For a 100 μL RCA solution, combine on ice        -   MIP probe-target DNA preparation (e.g., entire MIP            capture/cfDNA preparation described above, approximately 20            μL)        -   10 μL of 10× Phi29 Buffer for a 1× final concentration            -   1× Phi29 DNA Polymerase Reaction Buffer                -   50 mM Tris-HCl                -   10 mM MgCl₂                -   10 mM (NH₄)₂SO₄                -   4 mM DTT                -   (pH 7.5 @ 25° C.)            -   200 μM of each dNTP            -   5 units of Phi29 DNA polymerase            -   100 nM Beacon probe            -   molecular-grade water to 100 μL    -   Incubate 30° C. to 37° C. for reaction time, e.g., 90 to 120        minutes.        Enhanced RCA (E-RCA) Conditions:    -   For a 100 μL Enhanced RCA solution, combine on ice        -   MIP probe-target DNA preparation (e.g., entire MIP            capture/cfDNA preparation described above, approximately 20            μL);        -   10 μL of 10× Phi29 Buffer for a 1× final concentration        -   800 μM total dNTPs        -   80 units of Phi29 DNA polymerase        -   1000 nM Beacon probe        -   molecular-grade water to 100 μL    -   Incubate 30° C. for reaction time, e.g., 90 to 120 minutes.        One-Step Enhanced Rolling Circle Amplification on a Surface    -   Prepare Rolling Circle Amplification (RCA) solution        -   For a 100 μL RCA solution, combine on ice            -   MIP probe-target DNA preparation (e.g., entire MIP                capture/cfDNA preparation described above, approximately                20 μL);            -   10 μL of 10× Phi29 Buffer for a 1× final concentration                -   1× Phi29 DNA Polymerase Reaction Buffer                -    50 mM Tris-HCl                -    10 mM MgCl₂                -    10 mM (NH₄)₂SO₄                -    4 mM DTT                -    (pH 7.5 @ 25° C.)            -   4 μL of 10 mM or each dNTPs, for a 0.4 mM total dNTPs                final concentration;            -   50 μL of filtered 30% PEG 600;            -   0.5 μL of 100 μM Molecular Beacon for a final                concentration of 0.5 μM            -   2-8 μL of Phi29 polymerase (10 units/μL); and            -   22.5-28.5 μL of molecular-grade nuclease-free water        -   Mix solution, e.g., by vortexing, and pipet onto treated            glass surface comprising bound primers, then seal plate;        -   Incubate plate on flat bottom heat block of a thermomixer            with a thermo-lid, at 45° C. for 90 minutes;        -   Remove well contents and wash well two times with 100 μL of            1×TBS; discard wash solution;        -   Add 100 μL of 1×TBS, and image in microscope, as described            below.            Imaging Samples with IXM4 Microscope (Molecular Devices, San            Jose, Calif.)            Typically, 20×, 40×, or 60× objectives are used to capture            images.    -   Plates are placed in an IXM4 microscope (Molecular Devices,        Inc.) and imaged as follows:        -   Plates are auto-exposed to ensure a broad dynamic range            (maximum range of the camera used such as 16-bit images) in            the fluorescence intensity values.        -   Each well of the plate is sub-divided into approximately 100            images.            For high-throughput assays, automated microscopes may be            used.            Image Analysis    -   Images are analyzed as follows:        -   Relative fluorescence intensity was determined in images            containing no sample (negative control).        -   A threshold was determined by multiplying the average            relative fluorescent intensity from the negative control by            three.        -   Spots above the threshold using a localized threshold cutoff            calculated above are counted in each channel.            Variations on One-Step Protocol

Crowding reagent (e.g., PEG) addition: prepare a 30% solution inmolecular-grade water; filter with a 0.2 μm pore size filter. Add PEG tothe RCA reaction, adjusting the water added to the RCA to maintainconsistent volume.

Beacon: add desired concentration, adjusting the water added to the RCAto maintain consistent volume.

dNTPs: add desired concentration, adjusting the water added to the RCAto maintain consistent volume.

Detergents and detergent mixtures: Perform a 2-step reaction, adding adetergent or detergent mixture with the labeled probes. In someembodiments, a surface may be washed with a detergent mixture afteraddition of the labeled probes.

Intermittent mixing: 1-step or 2-step reaction protocols may includeintermittent mixing steps (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, etc. minutes) to re-distribute reagents at timepoints throughout the reaction step incubations (e.g., during probehybridization, rolling circle amplification, fluorescent probehybridization, etc.). Mixing is not limited to any particular method andmay comprising vortexing, bumping, rocking, tilting, or ultrasonicmixing. See, e.g., Ishigaki and Soto, Micromachines 2018, 9:272, whichis incorporated herein by reference for all purposes.

Graphene oxide: Perform a 2-step reaction, adding graphene oxide withthe labeled probes, as described in Example 2, below.

Example 2 Detection Using Two-Step Rolling Circle Amplification on aSurface

Prepare Rolling Circle Amplification (RCA) solution on ice:

-   -   For a 100 μL RCA solution (without molecular beacon), combine:        -   MIP probe-target DNA preparation (e.g., entire MIP            capture/cfDNA preparation described above, approximately 20            μL);        -   10 μL of 10× Phi29 Buffer for a 1× final concentration            -   1× Phi29 DNA Polymerase Reaction Buffer                -   50 mM Tris-HCl                -   10 mM MgCl₂                -   10 mM (NH₄)₂SO₄                -   4 mM DTT                -   (pH 7.5 @ 25° C.)        -   4 μL of 10 mM each dNTP, for a 0.4 mM total dNTPs final            concentration;        -   50 μL of filtered 30% PEG 600;        -   2-8 μL of Phi29 polymerase (10 units/μL); and        -   23 to 29 μL of molecular-grade water    -   Mix solution by vortexing and pipet onto treated glass surface,        then seal plate;    -   Incubate plate on flat bottom heat block of thermomixer with a        thermo-lid, at 45° C. for 90 minutes;    -   Remove well contents and wash well three times with 100 μL of        1×TBS; discard wash solution.        Staining Option 1    -   Add 50 μL of graphene oxide-molecular beacon solution that        comprises:        -   5 μL of 10× Phi 29 Buffer for a 1× final concentration        -   0.5 μL of 100 μM Molecular Beacon for a final concentration            of 0.5 μM        -   5 μL of 2 mg/mL graphene oxide solution for a final            concentration of 0.2 mg/mL;        -   Molecular-grade water to 50 μL    -   Incubate reaction for 60 minutes at 37° C.    -   Wash three times with 100 μL 1×TBS;    -   Wash one time with 100 μL 1×TBS containing 5% w:v Tween 20;    -   Wash two times with 100 μL of 1×TBS; discard wash solution;    -   Add 100 μL of 1×TBS, and image in microscope, as described        above.        Staining Option 2    -   Add:        -   5 μL of 10× Phi 29 Buffer for a 1× final concentration        -   0.5 μL of 100 μM Molecular Beacon or Fluorescent labeled            oligo for a final concentration of 0.5        -   5 μL of 0.1% Teepol detergent for a final concentration of            0.01% Teepol        -   Molecular-grade water to 50 μL    -   Incubate reaction for 30 minutes at 37° C.    -   Wash six times with 100 μL 1×TBS, and image in microscope, as        described above.

As discussed above, use of a 2-step protocol may be advantageous forcertain modifications, e.g., modifications that may be compatible withone step of the procedure but not compatible with another step.

Variations on the 2-Step Protocol

Intermittent mixing: 1-step or 2-step reaction protocols may includeintermittent mixing steps (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, etc. minutes) to re-distribute reagents at timepoints throughout the reaction step incubations (e.g., during probehybridization, rolling circle amplification, fluorescent probehybridization, etc.). Mixing is not limited to any particular method andmay comprising vortexing, bumping, rocking, tilting, or ultrasonicmixing. See, e.g., Ishigaki and Soto, Micromachines 2018, 9:272, whichis incorporated herein by reference for all purposes.

Example 3 Nuclease Digest to Remove Unligated MIP Probes

In some embodiments, ligated MIPs are treated with one or moreexonucleases to digest unligated probes. Two exemplary treatmentprotocols are described below. Reagents

New England Biolabs REAGENT Catalog Number 10 × CUTSMART Buffer B7204SThermolabile Exo I (20,000 U/mL) M0568S Rec Jf (30,000 U/mL) M0264S ExoVII (10,000 U/mL) M0379S Exo T (5,000 U/mL) M0265SProtocol 1

-   -   1. Use protocol below to prepare exo digest master mix:

Reagent ×1 (uL) Conc. 10 10 × Cutsmart Buffer 3 1× 30 Exo T (5 ,000U/mL) 0.75   15 U 7.5 Rec Jf (30,000 U/mL) 0.75 22.5 U 7.5 Nuclease FreeWater 0.50  7.5 U 7.5 Total Volume 5.00 52.5

-   -   2. Vortex master mix and pipet 5 μL of mix into each ligated        sample in PCR plate, for a total of 30 μL;    -   3. Foil seal PCR plate; vortex, then spin plate;    -   4. Put plate in thermocycler and follow the program below:

Exo program 37 C. 60 min 65 C. 30 min  4 C. HOLDProtocol 2

-   -   1. Use protocol below to prepare exo digest master mix:

Reagent ×1 (uL) Conc. 10 10 × Cutsmart Buffer 3 1× 30 Thermolabile Exo I(20,000 U/mL) 0.75   15 U 7.5 Rec Jf (30,000 U/mL) 0.75 22.5 U 7.5 ExoVII (10,000 U/mL) 0.75  7.5 U 7.5 Total Volume 5.25 52.5

-   -   2. Vortex master mix and pipet 5 μL of mix into each ligated        sample in PCR plate, for a total of 30.25 μL;    -   3. Foil seal PCR plate, vortex and spin plate;    -   4. Put plate in thermocycler and follow the program below:

Exo program 37° C. 60 min 80° C. 40 min  4° C. HOLD

The final high temperature step in each protocol heat denatures thenuclease and the solutions can then be used directly in RCA reactions.

Example 4 Preparation of Chemically Modified Assay Plates andOligonucleotide Primer Immobilization

Provided hereinbelow is an exemplary protocol for chemical modificationof assay plates using tannic acid or dopamine, and attachment ofoligonucleotides.

Polytannic Acid Plate Preparation Mfg: Catalog # Reagent Amount ScaleGreiner Bio-One: 655 892 96-well Microplate — Thermo Fisher: AC419995000Tannic Acid 8 mg Thermo Fisher: PI17874 Ammonium Persulfate 13 mg (APS)Thermo Fisher: PI17919 TEMED 17.2 μL Thermo Fisher: AA43359AP Acryl Acid550 μL H₂O to 10 mL 9.43 mL 15 mL Tube 1

-   -   1. Measure tannic acid and ammonium persulfate into 15 ml Tube        (or container)    -   2. Add nuclease-free H₂O to container    -   3. Add acrylic acid to solution    -   4. Add TEMED to solution    -   5. Vortex to mix    -   6. Pipette 504, to each well of the plate    -   7. Incubate at room temperature (25° C.) overnight (protected        from UV and other light)

Polydopamine Plate Preparation Mfg: Catalog # Reagent Amount ScaleGreiner Bio-One: 655 892 96-well Microplate — SIGMA: H8502 Dopamine 22mg Thermo Fisher: PI17874 Ammonium Persulfate (APS) 13 mg Thermo Fisher:PI17919 TEMED 17.2 μL SIGMA: 516155-25G 2- 1111 mgAminoethylmethacrylamide (AEMA) H₂O 9.43 mL 15 mL Tube 1

-   -   1. Measure AEMA into 15 ml Tube (or container)    -   2. Add nuclease free H₂O to container    -   3. Sonicate the container in a sonicating bath for 2 minutes    -   4. Centrifuge the container at 3500 rpm for 5 minutes    -   5. Aspirate the supernatant and filter through a 0.2 micron; if        needed, add more water to bring volume to 10 mL    -   6. Add the dopamine to the filtered solution    -   7. Add APS to solution    -   8. Add TEMED to solution    -   9. Vortex to mix    -   10. Pipette 504, to each well of the plate    -   11. Incubate at room temperature (25° C.) overnight (protected        from UV and other light)

Note: The following steps are identical for either tannic acid ordopamine plate preparation

Reagent removal and Washing Mfg: Catalog # Reagent Amount Scale (Any)Thermo Fisher: 10-977-023 H₂O 15 mL

-   -   1. Remove the tannic acid or dopamine solution from wells and        dispose into chemical waste    -   2. Wash the plate with nuclease-free water three (3) times using        100 μL per well. Remove the final wash fluid.    -   3. Allow plates to dry completely (e.g., overnight incubation at        room temperature (25° C.), or in a biological hood in Pre for at        least 3 hours before continuing to the oligo conjugation.

Conjugation of oligonucleotide to prepared assay plate Mfg: Catalog #Reagent Amount Scale IDT: Custom Custom Oligo (100 μM) 10 μL ThermoFisher: 22980 EDC (1-ethyl-3-(3- 98 mg dimethylaminopropyl)carbodiimidehydrochloride) Thermo Fisher: 24500 NHS (N-hydroxysuccinimide) 57 mg(Any) Thermo Fisher: 10-977-023 H₂O 9,970 μL + 2 mL (Any) Thermo Fisher:AM12500 15 mL Tube 1

-   -   1. Prepare a 100 mM solution of EDC by measuring out 98 mg of        EDC and adding 1 mL of H₂O.    -   2. Prepare a 100 mM solution of NHS by measuring out 57 mg of        NHS and adding 1 mL of H₂O.    -   3. Combine the following into a 15 ml conical tube (or        container)        -   a. 100 μL of 100 μM amine-modified oligonucleotide        -   b. 1000 μL of 100 mM EDC        -   c. 1000 μL of 100 mM NHS        -   d. 7900 μL of H₂O    -   4. Vortex to mix    -   5. Dispense 50 μL of this solution into each well of the plate    -   6. Incubate the plate at 37° C. for one (1) hour    -   7. Wash the plate with nuclease-free water three (3) times using        100 μL per well. Remove the final wash fluid.        Plate Quality Control (QC) Check    -   QC plate with standard RCA performed in triplicates of 10 fmol,        1 fmol, 0.1 fmol, and 0 fmol of standard one color input.    -   QC reads for dopamine plates should fall around 30,000 spots for        10 fmol, 10,000 spots for 1 fmol, 2000 spots for 0.1 fmol, and 1        spot or less for the no-target control.    -   QC reads for tannic acid plates should fall around 15,000 spots        for 10 fmol, 5,000 spots for 1 fmol, 1000 spots for 0.1 fmol,        and 1 spot or less for the no-target control.

An example of an amine-modified oligonucleotide for conjugation to asurface is as shown below:

(SEQ ID NO: 17) 5′UniAmM/CGTCGTAGGTCACTTAACATAGAG3′“5′ UniAmM” indicates a 5′ Uni-Link™ Amino Modifier (Integrated DNATechnologies, Inc.)

Oligonucleotide modifications suitable for immobilization in thetechnology are not limited to the modifications on the oligonucleotideshown above. For example, in some embodiments, a spacer is added to anoligonucleotide conjugated to a surface. While not limiting theembodiment to any particular mechanism or effect, an oligonucleotideseparated from a plate or bead surface by a spacer would be expected tobe further into solution, such that interactions with the supportsurface are minimized and hybridization of circularized MIPs isenhanced, yielding additional detectable spots.

In some embodiments, multiple amine moieties are added to theoligonucleotides, since a single amine may be in the form H₃N⁺, whichcontains a positive charge and may not efficiently react to the NHSester. By adding additional amine groups, the percentage ofoligonucleotides with at least one reactive H₂N is increased, ensuringthat a higher percentage of the oligonucleotides are conjugated to theplate during the conjugation reaction.

Oligonucleotides that include spacers and/or additional amino groupsinclude but are not limited to the examples shown below:

(SEQ ID NO: 18) 5′UniAmM/TTTTCGTCGTAGGTCACTTAACATAGAG3′ (SEQ ID NO: 19)5′UniAmM/T/iUniAmM/T/iUniAmM/T/iUniAmM/TCGTCGTAGGT  CACTTAACATAGAG3′(SEQ ID NO: 20) 5′UniAmM/T/iUniAmM/T/iUniAmM/T/iUniAmM/T/iSp18/iSp18/CGTCGTAGGTCACTTAACATAGAG3′ (SEQ ID NO: 21)5′UniAmM/T/iUniAmM/T/iUniAmM/T/iUniAmM/T/iSp18/iSp18/iSp18/iSp18/CGTCGTAGGTCACTTAACATAGAG3′ (SEQ ID NO: 22)5UniAmNA/TTTT/iSp18/iSp18/CGTCGTAGGTCACTTAACATAGA G3′“iUniAmM” indicates an internal Uni-Link™ Amino Modifier and “iSP18”indicates an internal “Spacer 18,” an 18-atom hexa-ethyleneglycolspacer, (Integrated DNA Technologies, Inc.)

Example 5 Rolling Circle Amplification on Iron Oxide Nanoparticles

In some embodiments, rolling circle amplification is performedessentially in solution, e.g., it is performed using primers conjugatedto small iron oxide nanoparticles suspended in solution. An exemplaryprocedure is described below:

A. Conjugation of Amine-Containing Oligonucleotides to FunctionalizedIron Oxide Nanoparticles

Starting material: Ocean Nanotech catalog number SHP-10-10, 10 nm ironoxide nanoparticles with carboxylic acid reactive groups, provided at 5mg/mL (Fe) in 2 mL deionized H₂O with 0.02% NaN₃, 4.3 nM nmole/mL ofnanoparticles. For each oligonucleotide, use 5 μL of the suspension, orabout 20 pmoles beads, for 1 pmole of oligonucleotide.

-   -   1. Magnetize or spin nanoparticles (standard desktop        microcentrifuge at 13,000 RPMs or 20,000 RCF for 5 minutes) and        remove storage buffer. Resuspend in 50 μL of nuclease-free        water.    -   2. Magnetize or spin nanoparticles as above and remove        supernatant. Resuspend in 50 microliters of a solution        containing 0.5 mM EDC and 0.5 mM NHS with 0.1 μM of an        amine-containing oligonucleotide.        -   a. Freshly prepare a 500 mM concentration of NHS and EDC in            nuclease-free water. Combine aliquots with an aliquot of            amine-containing oligonucleotide from a 100 μM stock to            produce the solution containing 0.5 mM EDC and 0.5 mM NHS            with 0.1 μM of an amine-containing oligonucleotide.    -   3. Incubate nanoparticle-oligonucleotide mixture at 37° C. for 1        hour.    -   4. Magnetize or spin the nanoparticles as above and remove the        supernatant without disrupting the pellet. Add 50 μL of        nuclease-free water.    -   5. Repeat step 4 three times. After the final wash resuspend the        nanoparticles in 25 μL of nuclease-free water.        Rolling Circle Amplification with Iron Oxide Nanoparticle-Bound        Primer Oligonucleotides        The following oligonucleotide was conjugated to iron oxide        nanoparticles as described above:

(SEQ ID NO: 35) 5'-/5UniArnM/CGTCGTAGGTCACTTAACATAGAGTT/3BioTEG/-3′Real-time qRCA reactions were performed with the following variations inreagent concentrations.

i. Circular Template DNA: 0, 100, 500, or 1000 fmol

ii. Nanoparticle-primer: 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM

iii. Molecular beacon probe: 0.25 μM, 0.5 μM, 5 μM, 10 μM, 15 μM, and 20μM

-   -   1. Prepare an RCA master mix as follows:

Master Mix Component For each reaction 10 × Phi29 Buffer 10 μL 10 mMeach dNTP mix  4 μL 30% PEG Molecular Weight 600 50 μL Input MIP 1A (200fmoles per μL)  5 μL Iron Oxide nanoparticle-primer 25 μL Phi29Polymerase (10 u per μL)  6 μL Total Vol 100 μL 

-   -   2. Incubated the RCA reactions at 45° C. for 90 minutes, with        at-temperature mixing at 1500 rmp for the first 30 seconds of        each 15 minute interval.    -   3. Magnetize or spin nanoparticles as described above and remove        the supernatant without disrupting the pellet. Resuspend the        nanoparticles in 50 μL of nuclease-free water.    -   4. Repeat Step 8 three times.    -   5. Hybridize the RCA products on the nanoparticles to        fluorophore-labeled oligonucleotides as described above. For        example:        -   a. Add 50 μL of molecular beacon solution that comprises:            -   i. 5 μL of 10× Phi 29 Buffer for a 1× final                concentration            -   ii. 0.5 μL of 100 μM Molecular Beacon for a final                concentration of 0.5 μM            -   iii. Molecular-grade water to 50 μL        -   b. Incubate reaction for 60 minutes at 37° C.        -   c. Magnetize or spin nanoparticles as described above and            remove the supernatant without disrupting the pellet.            Resuspend the nanoparticles in 50 μL 1×TBS;        -   d. Repeat step c. 2 additional times.        -   e. Wash nanoparticles one time with 50 μL 1×TBS containing            5% w:v Tween 20;    -   6. Wash two times with 50 μL of 1×TBS; as described above,        Magnetize or spin nanoparticles as described above and remove        the supernatant without disrupting the pellet. Resuspend the        nanoparticles in 50 μL of nuclease-free water.    -   7. Place the nanoparticle solution in a well of a 96 well plate        for imaging on a microscope. Magnetize or spin the nanoparticles        into a layer on the plate for visualization in the focal plane        of the microscope. Count fluorescing nanoparticles.

For an endpoint read as shown in FIG. 55, the assay plate is imageddirectly. For qRCA as shown in FIG. 54, reactions were incubated at 37°C. and the fluorescence was measured at the end of each minute for 720cycles (approximately 720 minutes).

Example 6 In-Solution Rolling Circle Amplification with Filter Capture

Padlock Probe Ligation

Cf DNA is treated by restriction endonuclease digestion. Followingdigestion, digested DNA is then combined with

-   -   5 pM each locus-specific probe    -   508 nM per backbone (21×10¹² molecules)    -   80 U of Taq DNA Ligase    -   1 mM NAD+    -   100 mM NaCl    -   15 mM MgCl₂    -   10 mM Tris-HCl pH 8 and 0.11% w/v Tween20

The reaction mixture was incubated at 95° C. for 5 minutes, followed by10 hours at 56° C. 39 μl of an exonuclease master mix consisting of 20 UExonuclease I, 5 U Lambda exonuclease, 100 U Exonuclease III, 10 UUracil dehydrogenase and 28% Tween20 was added to each reaction. Themixture was incubated at 37° C. for 1 hour, followed by 80° C. for 20minutes.

RCA Reaction

Following Exo III treatment, RCA was performed by adding 11.2 μl RCAmaster mix consisting of 1.07 μM of an RCA primer oligonucleotidedesigned to be complementary to a sequence present in the backboneregion of each DNA circle, 5.4 mM dNTP solution mix and 20 units ofPhi29 DNA polymerase. The reaction was incubated at 37° C. for 1 hour,followed by 65° C. for 10 minutes.

Probe Hybridization

Following RCA, a 12 μl labeling master mix consisting of 60 nM of eachfluorescent labeling oligonucleotide complementary to a chromosomal tagin the backbone, 12×SSC (saline sodium citrate) buffer and 0.6%Tween-20. The reaction was incubated at 45° C. for 1 hour. Each reactionwas then filtered through a well of a nanofilter detection plate(PerkinElmer), the plate comprising pores selected to retain the labeledRCA products on the upper membrane surface. The membrane was washedtwice in 0.5×SSC and the detection plate was moved to a blottingmembrane to remove residual liquid. After drying, an optical clearingagent (PerkinElmer) was added to each well and allowed to cure for 10minutes. The plates were imaged and analyzed using a Vanadis View™microplate scanner (PerkinElmer).

Example 7 In-Solution Rolling Circle Amplification with Plate Capture

In-sol primers 1, 2, 3, 4, 5, 6, and 7 as shown in FIG. 57A were testedin reactions in which RCA products were formed in solution and capturedon a plate for counting. The signal was compared to on-plate RCAperformed using on-plate oligonucleotides shown in FIG. 57B. Padlockprobe ligation reactions comprised the following:

[Final] Reagents ×1 ×10 1 × Amp Buffer 2.5 25 Water 1.75 17.5 1 × NAD0.25 2.5 1 uM MIP 1B 5 50 1 uM Target 1T 15 150 Ampligase (0.1 U/μL) 0.55 Total Volume 25 250

Ligation reactions were incubated at 98° C. for 3 min, then 45° C. 60min, then held at 4° C. until further processing. In some embodiments,the ligation mixture is treated with exonuclease to remove excess MIPprobe. Exonuclease treatment may be omitted when the MIP probes are notpresent in excess, e.g., when the concentration of target strands (e.g.,synthetic target strands) are at about the same concentration as the MIPprobes in the ligation mixture, as shown in the table above.

Quantitative RCA (qRCA) reactions comprised the following:

[Final] Reagents ×1 ×10 10 × Phi29 Buffer 2.5 25 Biotin Primer 1:100 2.525 water 13.25 132.5 dNTPs 1 10 100 uM Beacon 0.25 5 Phi29 Polymerase0.5 5 Ligated MIP reaction mix 5 50 Total Volume 25 250

Reactions were performed in triplicate for each primer type. qRCAreactions were incubated at 45° C. Endpoint results for the in-solutionRCA and the on-plate RCA are shown in FIG. 58.

RCA efficiency for the in-solution primers was also examined in astandard qRCA reaction, with the results shown FIG. 59, compared to qRCAprimed using the AUP control primer shown on FIG. 57B (performed insolution). The presence of 3′ biotin on the in-solution primers appearsto delay the reaction, resulting in approximately 2-fold less productthan that generated by the AUP control primer. For both types of primer,the majority of the RCA product is generated within the first hour TheRCA product is generated within the first hour of the RCA step, asindicated by the curve generated by 50 cycles/50 minutes in the qRCA(Note: RCA is performed at a single temperature and each “cycle” refersto a 1 minute interval at that temperature). These data also suggestthat the length of the RCA product generated by the current NeverseqPrimer (AUP) and the In-solution primers is similar.

Reaction efficiencies for in-solution qRCA performed as described abovewere examined using the following combinations of reactants:

-   -   1. 0, 100, 500 and 1000 fmol DNA input with 0.5 μM of primer,        with 0.25 μM beacon probe    -   2. 0, 100, 500 and 1000 fmol DNA input with 1 μM of primer, with        0.5 μM beacon probe    -   3. 0, 100, 500 and 1000 fmol DNA input with 5 μM of primer, with        5 μM beacon probe    -   4. 0, 100, 500 and 1000 fmol DNA input with 10 μM of primer,        with 10 μM beacon probe    -   5. 0, 100, 500 and 1000 fmol DNA input with 15 μM of primer,        with 15 μM beacon probe    -   6. 0, 100, 500 and 1000 fmol DNA input with 20 μM of amine        primer, with 20 μM beacon probe

Input DNA refers to circularized probe DNA. These reactions showed thatunder the conditions tested, increasing the concentrations of primer andbeacon probe increased the signal up to concentrations of 10 μM ofprimer and 10 μM beacon probe, above which signal did not increase (datanot shown).

It is readily apparent that, provided with the disclosure herein, eachof the front-end target recognition systems disclosed may be configuredto generate a signal detectable for use with any one of the back-endinstruments and systems described above.

ADDITIONAL MATERIALS INCORPORATED HEREIN BY REFERENCE

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All literature and similar materials cited in this application,including the publications described in the Bibliography above, andincluding but not limited to patents, patent applications, articles,books, treatises, and internet web pages, are expressly incorporated byreference in their entireties for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology, molecular diagnostics, nucleic acids structure,biochemistry, medical science, or related fields are intended to bewithin the scope of the claims.

We claim:
 1. A method for nucleic acid molecule analysis, comprising: a)providing a plurality of molecular inversion probes (MIPs) and a sampleof nucleic acid molecules; b) hybridizing MIPs of the plurality of MIPsto the nucleic acid molecules to generate hybridized MIPs; c) in areaction mixture, circularizing hybridized MIPs to form a plurality ofcircularized nucleic acid probes; d) treating the reaction mixture withmore than one exonuclease, wherein circularized nucleic acid probes arenot a substrate for the more than one exonucleases; e) forming aplurality of complexes comprising a plurality of primers hybridized to aplurality of circularized nucleic acid probes from the reaction mixturefollowing d), wherein the plurality of primers is bound to a solidsupport; f) extending the plurality of primers in the plurality ofcomplexes in an amplification reaction to form a plurality ofamplification products immobilized to the solid support; g) in apresence of a detergent, hybridizing a set of labeled probes to theplurality of amplification products to generate a plurality ofamplification products comprising hybridized labeled probes, wherein theset of labeled probes comprises five or more different labels; and h)using imaging to count the plurality of amplification productscomprising hybridized labeled probes based on detecting the five or moredifferent labels.
 2. The method of claim 1, wherein the more than oneexonucleases comprise Rec Jf.
 3. The method of claim 1, wherein the morethan one exonucleases comprise Exo VII.
 4. The method of claim 1,wherein the more than one exonucleases comprise Exo I.
 5. The method ofclaim 4, wherein the more than one exonucleases comprise ThermolabileExo I.
 6. The method of claim 1, wherein the more than one exonucleasescomprise Rec Jf, Exo VII, and Exo I.
 7. The method of claim 1, whereinthe more than one exonucleases comprise Rec Jf, Exo VII, andThermolabile Exo I.
 8. The method of claim 1, further comprising,between d) and e), inactivating the more than one exonucleases.
 9. Themethod of claim 8, wherein the inactivating comprises heat-inactivating.10. The method of claim 1, wherein the solid support comprises tannicacid.
 11. The method of claim 1, wherein the solid support comprisesacrylic acid.
 12. The method of claim 1, wherein the solid supportcomprises a homopolymeric coating.
 13. The method of claim 1, whereinthe plurality of primers is bound to the solid support by a 5′ terminalamine modification.
 14. The method of claim 1, wherein the plurality ofprimers is covalently bound to the solid support.
 15. The method ofclaim 1, wherein the solid support comprises glass.
 16. The method ofclaim 1, wherein the solid support comprises an assay plate.
 17. Themethod of claim 16, wherein the assay plate is a multi-well assay plate.18. The method of claim 1, wherein in f) the plurality of amplificationproducts immobilized to the solid support are in a solution comprising acrowding agent.
 19. The method of claim 18, wherein the crowding agentcomprises polyethylene glycol (PEG).
 20. The method of claim 19, whereinthe PEG has an average molecular weight between 200 and
 8000. 21. Themethod of claim 19, wherein the PEG is at a concentration of at least12%.
 22. The method of claim 1, wherein the plurality of primers arebound to the solid support in an irregular dispersal.
 23. The method ofclaim 1, wherein the five or more different labels comprise five or moredifferent fluorescent labels.